EP3645728A1 - Nouveaux orthologues de crispr de type vi et systèmes associés - Google Patents

Nouveaux orthologues de crispr de type vi et systèmes associés

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Publication number
EP3645728A1
EP3645728A1 EP18824952.8A EP18824952A EP3645728A1 EP 3645728 A1 EP3645728 A1 EP 3645728A1 EP 18824952 A EP18824952 A EP 18824952A EP 3645728 A1 EP3645728 A1 EP 3645728A1
Authority
EP
European Patent Office
Prior art keywords
cell
rna
sequence
guide
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18824952.8A
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German (de)
English (en)
Other versions
EP3645728A4 (fr
Inventor
Feng Zhang
Omar O. Abudayyeh
David Benjamin Turitz Cox
Jonathan S. Gootenberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
Massachusetts Institute of Technology
Broad Institute Inc
Original Assignee
Harvard College
Massachusetts Institute of Technology
Broad Institute Inc
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Publication date
Application filed by Harvard College, Massachusetts Institute of Technology, Broad Institute Inc filed Critical Harvard College
Publication of EP3645728A1 publication Critical patent/EP3645728A1/fr
Publication of EP3645728A4 publication Critical patent/EP3645728A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the subject matter disclosed herein is generally directed to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as perturbation of gene transcripts or nucleic acid editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture.
  • the CRISPR-Cas system loci has more than 50 gene families and there is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture.
  • CRISPR-Cas systems A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multisubunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein.
  • Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important.
  • the CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference.
  • the Class 2 type VI single-component CRISPR-Cas effector C2c2 (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) was characterized as an RNA-guided Rnase (Abudayyeh et al.
  • C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”; doi: 10.1126/science.aaf5573). It was demonstrated that C2c2 (e.g. from Leptotrichia shahii) that provides robust interference against RNA phage infection. Through in vitro biochemical analysis and in vivo assays, it was shown that C2c2 can be programmed to cleave ssRNA targets carrying protospacers flanked by a 3' H (non-G) PAM.
  • Cleavage is mediated by catalytic residues in the two conserved HEPN domains of C2c2, mutations in which generate a catalytically inactive RNA-binding protein.
  • C2c2 is guided by a single guide and can be re-programmed to deplete specific mRNAs in vivo. It was shown that LshC2c2 can be targeted to a specific site of interest and can carry out non-specific RNase activity once primed with the cognate target RNA.
  • C2c2 is now known as Casl3a. It will be understood that the term “C2c2” herein is used interchangeably with “Cas 13 a”. [0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
  • RNA-targeting systems of the present application may transform the study and perturbation or editing of specific target sites through direct detection, analysis and manipulation, in particular in eukaryotic systems, more in particular in mammalian systems (including cells, organs, tissues, or organisms) and plant systems.
  • RNA-targeting systems of the present application effectively for RNA targeting without deleterious effects, it is critical to understand aspects of engineering and optimization of these RNA targeting tools.
  • the CRISPR-Casl3 family was discovered by computational mining of bacterial genomes for signatures of CRISPR systems (Shmakov, S. et al Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell 60, 385-397, doi: 10.1016/j.molcel.2015.10.008 (2015)), revealing the single-effector RNA-guided RNase Casl3a/C2c2 (Abudayyeh, O. O. et al C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.
  • the Class 2 type VI effector protein C2c2 also known as Casl3a, is a RNA-guided RNase that can be efficiently programmed to degrade ssRNA.
  • C2c2 (Casl3a) achieves RNA cleavage through conserved basic residues within its two HEPN domains, in contrast to the catalytic mechanisms of other known RNases found in CRISPR-Cas systems.
  • Mutation of the HEPN domain such as (e.g. alanine) substitution, at any of the four predicted HEPN domain catalytic residues converted C2c2 into an inactive programmable RNA-binding protein (dC2c2, analogous to dCas9).
  • RNA-guided RNase Casl3 would make it an ideal platform for transcriptome manipulation.
  • Applicants herein develop Casl3c for use as a mammalian transcript knockdown and binding tool.
  • Casl3c is capable of robust RNA cleavage and binding with catalytically inactive versions using programmable guides.
  • dCasl3c to bind to specified sequences could be used in several aspects according to the invention to (i) bring effector modules to specific transcripts to modulate the function or translation, which could be used for large-scale screening, construction of synthetic regulatory circuits and other purposes; (ii) fluorescently tag specific RNAs to visualize their trafficking and/or localization; (iii) alter RNA localization through domains with affinity for specific subcellular compartments; and (iv) capture specific transcripts (through direct pull down of dCasl3c or use of dCasl3c to localize biotin ligase activity to specific transcripts) to enrich for proximal molecular partners, including RNAs and proteins.
  • Active Casl3c should also have many applications.
  • An aspect of the invention involves targeting a specific transcript for destruction, as with RFP here.
  • Cas 13c once primed by the cognate target, may cleave other (non-complementary) RNA molecules in vitro and can inhibit cell growth in vivo.
  • Biologically, such promiscuous RNase activity may reflect a programmed cell death/dormancy (PCD/D)-based protection mechanism of the type VI CRISPR- Cas systems.
  • PCD/D programmed cell death/dormancy
  • PCD or dormancy in specific cells—for example, cancer cells expressing a particular transcript, neurons of a given class, cells infected by a specific pathogen, or other aberrant cells or cells the presence of which is otherwise undesirable.
  • specific cells for example, cancer cells expressing a particular transcript, neurons of a given class, cells infected by a specific pathogen, or other aberrant cells or cells the presence of which is otherwise undesirable.
  • the invention provides a method of modifying nucleic acid sequences associated with or at a target locus of interest, in particular in eukaryotic cells, tissues, organs, or organisms, more in particular in mammalian cells, tissues, organs, or organisms, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Type VI CRISPR- Cas loci effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest.
  • the modification is the introduction of a strand break.
  • sequences associated with or at the target locus of interest comprises RNA and the effector protein is encoded by a type VI CRISPR- Cas loci.
  • the complex can be formed in vitro or ex vivo and introduced into a cell or contacted with RNA; or can be formed in vivo.
  • Cas enzyme CRISPR enzyme
  • CRISPR protein Cas protein CRISPR Cas
  • CRISPR Cas CRISPR Cas
  • the invention provides a method of targeting (such as modifying) sequences associated with or at a target locus of interest, the method comprising delivering to said sequences associated with or at the locus a non-naturally occurring or engineered composition comprising a Casl3c loci effector protein (which may be catalytically active, or alternatively catalytically inactive) and one or more nucleic acid components, wherein the Casl3c effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of sequences associated with or at the target locus of interest.
  • the modification is the introduction of a strand break.
  • the Casl3c effector protein forms a complex with one nucleic acid component; advantageously an engineered or non-naturally occurring nucleic acid component.
  • the complex can be formed in vitro or ex vivo and introduced into a cell or contacted with RNA; or can be formed in vivo.
  • the induction of modification of sequences associated with or at the target locus of interest can be Casl3c effector protein-nucleic acid guided.
  • the one nucleic acid component is a CRISPR RNA (crRNA).
  • 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.
  • the spacer sequence or the derivative thereof comprises a seed sequence, wherein the seed sequence is critical for recognition and/or hybridization to the sequence at the target locus.
  • the nucleic acid component of the complex may comprise a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem loops or optimized secondary structures.
  • the direct repeat has a minimum length of 16 nts, such as at least 28 nt, and a single stem loop.
  • the direct repeat has a length longer than 16 nts, preferably more than 17 nts, such as at least 28 nt, and has more than one stem loop or optimized secondary structures.
  • the direct repeat has 25 or more nts, such as 26 nt, 27 nt, 28 nt or more, and one or more stem loop structures.
  • the direct repeat may be modified to comprise one or more protein-binding RNA aptamers.
  • one or more aptamers may be included such as part of optimized secondary structure. Such aptamers may be capable of binding a bacteriophage coat protein.
  • the bacteriophage coat protein may be selected from the group comprising Qp, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, L95, TW19, AP205, ⁇ )5, ( ⁇ Cb8r, ( ⁇ Cbl2r, ( ⁇ Cb23r, 7s and PRR1.
  • the bacteriophage coat protein is MS2.
  • the invention also provides for the nucleic acid component of the complex being 30 or more, 40 or more or 50 or more nucleotides in length.
  • the invention provides cells comprising the type VI effector protein and/or guides and or complexes thereof with target nucleic acids.
  • 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.
  • the invention also provides a method of modifying a target locus of interest, in particular in eukaryotic cells, tissues, organs, or organisms, more in particular in mammalian cells, tissues, organs, or organisms, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Casl3c loci effector protein and one or more nucleic acid components, wherein the Casl3c effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the target locus of interest.
  • the modification is the introduction of a strand break.
  • the complex can be formed in vitro or ex vivo and introduced into a cell or contacted with RNA; or can be formed in vivo.
  • the target locus of interest may be comprised within an RNA moledule.
  • the target locus of interest may be comprised within a DNA molecule, and in certain embodiments, within a transcribed DNA molecule.
  • the target locus of interest may be comprised in a nucleic acid molecule in vitro.
  • the target locus of interest may be comprised in a nucleic acid molecule within a cell, in particular a eukaryotic cell, such as a mammalian cell or a plant cell.
  • the mammalian cell many be a non-human primate, bovine, porcine, rodent or mouse cell.
  • the cell may be a non-mammalian eukaryotic cell such as poultry, fish or shrimp.
  • the plant cell may be of a crop plant such as cassava, corn, sorghum, wheat, or rice.
  • the plant cell may also be of an algae, tree or vegetable.
  • the modification introduced to the cell by the present invention may be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output.
  • the modification introduced to the cell by the present invention may be such that the cell and progeny of the cell include an alteration that changes the biologic product produced.
  • the mammalian cell many be a non-human mammal, e.g., primate, bovine, ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mouse cell.
  • the cell may be a non-mammalian eukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell.
  • the cell may also be a plant cell.
  • the plant cell may be of a monocot or dicot or of a crop or grain plant such as cassava, corn, sorghum, soybean, wheat, oat or rice.
  • the plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus Spinacia; plants of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).
  • fruit or vegetable e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the gen
  • the invention provides a method of modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Type VI CRISPR-Cas loci effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the target locus of interest.
  • the modification is the introduction of a strand break.
  • the invention also provides a method of modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Casl3c loci effector protein and one or more nucleic acid components, wherein the Casl3c effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the target locus of interest.
  • the modification is the introduction of a strand break.
  • the target locus of interest may be comprised in a nucleic acid molecule in vitro.
  • the target locus of interest may be comprised in a nucleic acid molecule within a cell.
  • the target locus of interest may be comprised in a RNA molecule in vitro.
  • the target locus of interest may be comprised in a RNA molecule within a cell.
  • the cell may be a prokaryotic cell or a eukaryotic cell.
  • the cell may be a mammalian cell.
  • the cell may be a rodent cell.
  • the cell may be a mouse cell.
  • the target locus of interest may be a genomic or epigenomic locus of interest.
  • the complex may be delivered with multiple guides for multiplexed use.
  • more than one protein(s) may be used.
  • the nucleic acid components may comprise a CRISPR RNA (crRNA) sequence.
  • crRNA CRISPR RNA
  • the pre-crRNA may comprise secondary structure that is sufficient for processing to yield the mature crRNA as well as crRNA loading onto the effector protein.
  • such secondary structure may comprise, consist essentially of or consist of a stem loop within the pre-crRNA, more particularly within the direct repeat.
  • the effector protein and nucleic acid components may be provided via one or more polynucleotide molecules encoding the protein and/or nucleic acid component(s), and wherein the one or more polynucleotide molecules are operably configured to express the protein and/or the nucleic acid component(s).
  • the one or more polynucleotide molecules may comprise one or more regulatory elements operably configured to express the protein and/or the nucleic acid component(s).
  • the one or more polynucleotide molecules may be comprised within one or more vectors.
  • the target locus of interest may be a genomic or epigenomic locus of interest.
  • the complex may be delivered with multiple guides for multiplexed use.
  • more than one protein(s) may be used.
  • Regulatory elements may comprise inducible promotors.
  • Polynucleotides and/or vector systems may comprise inducible systems.
  • the one or more polynucleotide molecules may be comprised in a delivery system, or the one or more vectors may be comprised in a delivery system.
  • non-naturally occurring or engineered composition may be delivered via liposomes, particles including nanoparticles, exosomes, microvesicles, a gene-gun or one or more viral vectors.
  • the invention also provides a non-naturally occurring or engineered composition which is a composition having the characteristics as discussed herein or defined in any of the herein described methods.
  • the invention thus provides a non-naturally occurring or engineered composition, such as particularly a composition capable of or configured to modify a target locus of interest, said composition comprising a Type VI CRISPR-Cas loci effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the target locus of interest.
  • the effector protein may be a Casl3c loci effector protein.
  • the invention also provides in a further aspect a non-naturally occurring or engineered composition, such as particularly a composition capable of or configured to modify a target locus of interest, said 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, such as for multiplexing) or a nucleic acid encoding the guide RNA molecule (or one or more nucleic acids encoding the combination of guide RNA molecules); (b) a Type VI CRISPR-Cas loci effector protein or a nucleic acid encoding the Type VI CRISPR-Cas loci effector protein.
  • the effector protein may be a Casl3c loci effector protein.
  • the invention also provides in a further aspect a non-naturally occurring or engineered composition
  • a guide RNA molecule or a combination of guide RNA molecules, e.g., a first guide RNA molecule and a second guide RNA molecule
  • a nucleic acid encoding the guide RNA molecule or one or more nucleic acids encoding the combination of guide RNA molecules
  • (b) be a Casl3c loci effector protein 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 the guide RNA molecule (or one or more nucleic acids encoding the combination of guide RNA molecules); (b) be a Casl3c loci effector protein.
  • the invention also provides a vector system comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components of a non- naturally occurring or engineered composition which is a composition having the characteristics as defined in any of the herein described methods.
  • the invention also provides a delivery system comprising one or more vectors or one or more polynucleotide molecules, the one or more vectors or polynucleotide molecules comprising one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition which is a composition having the characteristics discussed herein or as defined in any of the herein described methods.
  • the invention also provides a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or vector or delivery systems comprising one or more polynucleotides encoding components of said composition for use in a therapeutic method of treatment.
  • the therapeutic method of treatment may comprise gene or transcriptome editing, or gene therapy.
  • the invention also provides for methods and compositions wherein one or more amino acid residues of the effector protein may be modified e.g., an engineered or non-naturally- occurring effector protein or Casl3c.
  • the modification may comprise mutation of one or more amino acid residues of the effector protein.
  • the one or more mutations may be in one or more catalytically active domains of the effector protein.
  • the effector protein may have reduced or abolished nuclease activity compared with an effector protein lacking said one or more mutations.
  • the effector protein may not direct cleavage of the RNA strand at the target locus of interest.
  • the one or more mutations may comprise two mutations.
  • the one or more amino acid residues are modified in a Casl3c effector protein, e.g., an engineered or non-naturally-occurring effector protein or Casl3c.
  • the one or more modified or mutated amino acid residues are one or more of those in C2c2 corresponding to R597, H602, R1278 and H1283 (referenced to Lsh C2c2 amino acids), such as mutations R597A, H602A, R1278A and H1283A, or the corresponding amino acid residues in Lsh C2c2 orthologues.
  • the one or more modified of mutated amino acid residues are one or more of those in C2c2 corresponding to K2, K39, V40, E479, L514, V518, N524, G534, K535, E580, L597, V602, D630, F676, L709, 1713, R717 (HEPN), N718, H722 (HEPN), E773, P823, V828, 1879, Y880, F884, Y997, L1001, F1009, L1013, Y1093, L1099, LI 111, Y1114, L1203, D1222, Y1244, L1250, L1253, K1261, 11334, L1355, L1359, R1362, Y1366, E1371, R1372, D1373, R1509 (HEPN), H1514 (HEPN), Y1543, D1544, K1546, K1548, V1551, 11558, according to C2c2 consensus numbering.
  • the one or more modified of mutated amino acid residues are one or more of those in C2c2 corresponding to R717 and R1509. In certain embodiments, the one or more modified of mutated amino acid residues are one or more of those in C2c2 corresponding to K2, K39, K535, K1261, R1362, R1372, K1546 and K1548. In certain embodiments, said mutations result in a protein having an altered or modified activity. In certain embodiments, said mutations result in a protein having an increased activity, such as an increased specificity. In certain embodiments, said mutations result in a protein having a reduced activity, such as reduced specificity. In certain embodiments, said mutations result in a protein having no catalytic activity (i.e. "dead" C2c2). In an embodiment, said amino acid residues correspond to Lsh C2c2 amino acid residues, or the corresponding amino acid residues of a C2c2 protein from a different species.
  • the one or more modified of mutated amino acid residues are one or more of those in C2c2 corresponding to M35, K36, T38, K39, 157, E65, G66, L68, N84, T86, E88, 1103, N105, E123, R128, R129, K139, L152, L194, N196, K198, N201, Y222, D253, 1266, F267, S280, 1303, N306, R331, Y338, K389, Y390, K391, 1434, K435, L458, D459, E462, L463, 1478, E479, K494, R495, N498, S501, E519, N524, Y529, V530, G534, K535, Y539, T549, D551, R577, E580, A581, F582, 1587, A593, L597, 1601, L602, E611, E613, D630, 1631, G63
  • the one or more modified of mutated amino acid residues are one or more conserved charged amino acid residues.
  • said amino acid residues may be mutated to alanine.
  • the one or more modified of mutated amino acid residues are one or more of those in C2c2 corresponding to K28, K31, R44, E162, E184, K262, E288, K357, E360, K338, R441 (HEPN), H446 (HEPN), E471, K482, K525, K558, D707, R790, K811, R833, E839, R885, E894, R895, D896, K942, R960 (HEPN), H965 (HEPN), D990, K992, K994 with reference to the consensus sequence as indicated in Figure 2, i.e. based on the alignment of the C2c2 orthologues as indicated in Figure 1.
  • the residues corresponding to R597, H602, R1278 and H1283 are excluded.
  • the invention also provides for the one or more mutations or the two or more mutations to be in a catalytically active domain of the effector protein.
  • the one or more mutations or the two or more mutations may be in a catalytically active domain of the effector protein comprising a HEPN domain, or a catalytically active domain which is homologous to a HEPN domain.
  • 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 domain(s) may be positioned at or near or in proximity to a terminus of the effector protein (e.g., Casl3c) and if two or more NLSs, each of the two may be positioned at or near or in proximity to a terminus of the effector protein (e.g., Casl3c).
  • the one or more heterologous functional domains may comprise one or more translational activation domains.
  • the functional domain may comprise a transcriptional activation domain, for example VP64.
  • the one or more heterologous functional domains may comprise one or more transcriptional repression domains.
  • the transcriptional repression domain comprises a KRAB domain or a SID domain (e.g. SID4X).
  • the one or more heterologous functional domains may comprise one or more nuclease domains.
  • a nuclease domain comprises Fokl .
  • the invention also provides for the one or more heterologous functional domains to have one or more of the following activities: methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity.
  • the one or more heterologous functional domains may comprise epitope tags or reporters.
  • Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporters include, but are not limited to, glutathione-S- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta- galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S- transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta- galactosidase beta- galactos
  • At least one or more heterologous functional domains may be at or near the amino- terminus of the effector protein and/or wherein at least one or more heterologous functional domains is 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.
  • the invention also provides for the effector protein comprising an effector protein from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylo
  • the effector protein may comprise a chimeric effector protein comprising a first fragment from a first effector protein ortholog and a second fragment from a second effector protein ortholog, and wherein the first and second effector protein orthologs are different.
  • At least one of the first and second effector protein orthologs may comprise an effector protein from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes
  • the effector protein may originate from, may be isolated from, or may be derived from a bacterial species belonging to the taxa alpha-proteobacteria, Bacilli, Clostridia, Fusobacteria and Bacteroidetes.
  • the effector protein may originate from, may be isolated from, or may be derived from a bacterial species belonging to a genus selected from the group consisting of Lachnospiraceae, Clostridium, Carnobacterium, Paludibacter, Listeria, Leptotrichia, and Rhodobacter.
  • the effector protein particularly a Type VI loci effector protein, more particularly a Casl3cp may originate from, may be isolated from or may be derived from a bacterial species selected from the group consisting of Lachnospiraceae bacterium MA2020, Lachnospiraceae bacterium NK4A179, Clostridium aminophilum (e.g., DSM 10710), Lachnospiraceae bacterium K4A144, Carnobacterium gallinarum (e.g., DSM 4847 strain MT44), Paludibacter propionicigenes (e.g., WB4), Listeria seeligeri (e.g., serovar 1 ⁇ 2b str.
  • a Type VI loci effector protein more particularly a Casl3cp may originate from, may be isolated from or may be derived from a bacterial species selected from the group consisting of Lachnospiraceae bacterium MA2020, Lachnospiraceae bacterium NK4A179, Clo
  • SLCC3954 Listeria weihenstephanensis (e.g., FSL R9-0317 c4), Listeria newyorkensis (e.g., strain FSL M6-0635: also "LbFSL”), Leptotrichia wadei (e.g., F0279: also "Lw” or "Lw2”), Leptotrichia buccalis (e.g., DSM 1135), Leptotrichia sp. Oral taxon 225 (e.g., str. F0581), Leptotrichia sp.
  • LbFSL Listeria newyorkensis
  • Leptotrichia wadei e.g., F0279: also "Lw” or “Lw2”
  • Leptotrichia buccalis e.g., DSM 1135
  • Oral taxon 225 e.g., str. F0581
  • Oral taxon 879 e.g., strain F0557
  • Leptotrichia shahii e.g., DSM 19757
  • Rhodobacter capsulatus e.g., SB 1003, R121, or DE442.
  • the Casl3c effector protein originates from Listeriaceae bacterium (e.g.
  • FSL M6-0635 also "LbFSL”
  • Lachnospiraceae bacterium MA2020 Lachnospiraceae bacterium MA2020
  • Lachnospiraceae bacterium NK4A179 Clostridium aminophilum (e.g., DSM 10710)
  • Carnobacterium gallinarum e.g., DSM 4847
  • Paludibacter propionicigenes e.g., WB4
  • Listeria seeligeri e.g., serovar 1 ⁇ 2b str.
  • a Type VI locus as intended herein may encode Casl, Cas2, and the Casl3cp effector protein.
  • the effector protein particularly a Type VI loci effector protein, more particularly a Casl3cp, such as a native Casl3cp
  • the effector protein may be about 1000 to about 1500 amino acids long, such as about 1100 to about 1400 amino acids long, e.g., about 1000 to about 1100, about 1100 to about 1200 amino acids long, or about 1200 to about 1300 amino acids long, or about 1300 to about 1400 amino acids long, or about 1400 to about 1500 amino acids long, e.g., about 1000, about 1100, about 1200, about 1300, about 1400 or about 1500 amino acids long.
  • the effector protein particularly a Type VI loci effector protein, more particularly a Casl3cp, comprises at least one and preferably at least two, such as more preferably exactly two, conserved RxxxxH motifs. Catalytic RxxxxH motifs are are characteristic of HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains.
  • the effector protein, particularly a Type VI loci effector protein, more particularly a Casl3cp comprises at least one and preferably at least two, such as more preferably exactly two, HEPN domains.
  • the HEPN domains may possess RNAse activity.
  • the HEPN domains may possess DNAse activity.
  • Type VI loci as intended herein may comprise CRISPR repeats between 30 and 40 bp long, more typically between 35 and 39 bp long, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.
  • the direct repeat is at least 25 nt long.
  • a protospacer adj acent motif (PAM) or P AM-like motif directs binding of the effector protein complex as disclosed herein to the target locus of interest.
  • the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer).
  • the PAM may be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).
  • PAM may be used interchangeably with the term “PFS” or "protospacer flanking site” or “protospacer flanking sequence”.
  • the CRISPR enzyme is engineered and can comprise one or more mutations that reduce or eliminate a nuclease activity. Mutations can also be made at neighboring residues, e.g., at amino acids near those indicated above that participate in the nuclease activity. In some embodiments, only one HEPN domain is inactivated, and in other embodiments, a second HEPN domain is inactivated.
  • the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence and a guide sequence or spacer sequence.
  • 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.
  • the guide RNA or mature crRNA comprises 19 nts of partial direct repeat followed by 18, 19, 20, 21, 22, 23, 24, 25, or more nt of guide sequence, such as 18-25, 19-25, 20- 25, 21-25, 22-25, or 23-25 nt of guide sequence or spacer sequence.
  • the effector protein is a Casl3c effector protein and requires at least 16 nt of guide sequence to achieve detectable DNA cleavage and a minimum of 17 nt of guide sequence to achieve efficient DNA cleavage in vitro.
  • the effector protein is a Casl3c protein and requires at least 19 nt of guide sequence to achieve detectable RNA cleavage.
  • the direct repeat sequence is located upstream (i.e., 5') from the guide sequence or spacer sequence.
  • the seed sequence (i.e. the sequence essential critical for recognition and/or hybridization to the sequence at the target locus) of the Casl3c guide RNA is approximately within the first 5 nt on the 5' end of the guide sequence or spacer sequence.
  • the mature crRNA comprises a stem loop or an optimized stem loop structure or an optimized secondary structure.
  • the mature crRNA comprises a stem loop or an optimized stem loop structure in the direct repeat sequence, wherein the stem loop or optimized stem loop structure is important for cleavage activity.
  • the mature crRNA preferably comprises a single stem loop.
  • the direct repeat sequence preferably comprises a single stem loop.
  • the cleavage activity of the effector protein complex is modified by introducing mutations that affect the stem loop RNA duplex structure.
  • mutations which maintain the RNA duplex of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is maintained.
  • mutations which disrupt the RNA duplex structure of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is completely abolished.
  • the mature crRNA comprises a stem loop or an optimized stem loop structure.
  • the direct repeat of the crRNA comprises at least 25 nucleotides comprising a stem loop.
  • the stem is amenable to individual base swaps but activity is disrupted by most secondary structure changes or truncation of the crRNA. Examples of disrupting mutations include swapping of more than two of the stem nucleotides, addition of a non-pairing nucleotide in the stem, shortening of the stem (by removal of one of the pairing nucleotides) or extending the stem (by addition of one set of pairing nucleotides).
  • the crRNA may be amenable to 5' and/or 3' extensions to include nonfunctional RNA sequences as envisaged for particular applications described herein.
  • the invention also provides for the nucleotide sequence encoding the effector protein being codon optimized for expression in a eukaryote or eukaryotic cell in any of the herein described methods or compositions.
  • the codon optimized nucleotide sequence encoding the effector protein encodes any Casl3c discussed herein and is codon optimized for operability in a eukaryotic cell or organism, e.g., such cell or organism as elsewhere herein mentioned, for instance, without limitation, a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a human cell or non-human eukaryote organism, e.g., plant.
  • At least one nuclear localization signal is attached to the nucleic acid sequences encoding the Casl3c effector proteins.
  • at least one or more C-terminal or N-terminal NLSs are attached (and hence nucleic acid molecule(s) coding for the Casl3c effector protein can include coding for NLS(s) so that the expressed product has the NLS(s) attached or connected).
  • at least one nuclear export signal is attached to the nucleic acid sequences encoding the Casl3c effector proteins.
  • At least one or more C-terminal or N-terminal NESs are attached (and hence nucleic acid molecule(s) coding for the Casl3c effector protein can include coding for NES(s) so that the expressed product has the NES(s) attached or connected).
  • a C-terminal and/or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells.
  • the codon optimized effector protein is Casl3c and the spacer length of the guide RNA is from 15 to 35 nt.
  • 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.
  • the spacer length is from 15 to 17 nt, from 17 to 20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30 nt, from 30-35 nt, or 35 nt or longer.
  • the codon optimized effector protein is Casl3c and the direct repeat length of the guide RNA is at least 16 nucleotides. In certain embodiments, the codon optimized effector protein is Casl3c and the direct repeat length of the guide RNA is from 16 to 20 nt, e.g., 16, 17, 18, 19, or 20 nucleotides. In certain preferred embodiments, the direct repeat length of the guide RNA is 19 nucleotides.
  • the invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest.
  • the nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers.
  • the one or more aptamers may be capable of binding a bacteriophage coat protein.
  • the bacteriophage coat protein may be selected from the group comprising Qp, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ )5, ( ⁇ Cb8r, ( ⁇ Cbl2r, ( ⁇ Cb23r, 7s and PRR1.
  • the bacteriophage coat protein is MS2.
  • the invention also provides for the nucleic acid component of the complex being 30 or more, 40 or more or 50 or more nucleotides in length.
  • the invention provides a eukaryotic cell comprising a nucleotide sequence encoding the CRISPR system described herein which ensures the generation of a modified target locus of interest, wherein the target locus of interest is modified according to in any of the herein described methods.
  • a further aspect provides a cell line of said cell.
  • Another aspect provides a multicellular organism comprising one or more said cells.
  • the modification of the target locus of interest may result in: the eukaryotic cell comprising altered (protein) expression of at least one gene product; the eukaryotic cell comprising altered (protein) expression of at least one gene product, wherein the (protein) expression of the at least one gene product is increased; the eukaryotic cell comprising altered (protein) expression of at least one gene product, wherein the (protein) expression of the at least one gene product is decreased; or the eukaryotic cell comprising an edited transcriptome.
  • the eukaryotic cell may be a mammalian cell or a human cell.
  • the non-naturally occurring or engineered compositions, the vector systems, or the delivery systems as described in the present specification may be used for RNA sequence-specific interference, RNA sequence specific modulation of expression (inluding isoform specific expression), stability, localization, functionality (e.g. ribosomal RNAs or miRNAs), etc.; or multiplexing of such processes.
  • the non-naturally occurring or engineered compositions, the vector systems, or the delivery systems as described in the present specification may be used for RNA detection and/or quantification in a sample, such as a biological sample.
  • RNA detection is in a cell.
  • the invention provides a method of detecting a target RNA in a sample, comprising (a) incubating the sample with i) a Type VI CRISPR-Cas effector protein capable of cleaving RNA, ii) a guide RNA capable of hybridizing to the target RNA, and iii) an RNA-based cleavage inducible reporter capable of being non- specifically and detectably cleaved by the effector protein, (b) detecting said target RNA based on the signal generated by cleavage of said RNA-based cleavage inducible reporter.
  • the Type VI CRISPR-Cas effector protein comprises a Casl3a effector protein. In an embodiment the Type VI CRISPR-Cas effector protein comprises a Casl3b effector protein. In an embodiment the Type VI CRISPR-Cas effector protein comprises a Casl3c effector protein. In an embodiment, the RNA-based cleavage inducible reporter construct comprises a fluorochrome and a quencher. In certain embodiments, the sample comprises a cell- free biological sample. In other embodiments, the sample comprises or a cellular sample, for example, without limitation a plant cell, or an animal cell.
  • the target RNA comprises a pathogen RNA, including, but not limited to a target RNA from a virus, bacteria, fungus, or parasite.
  • the guide RNA is designed to detect a target RNA which comprises a single nucleotide polymorphism or a splice variant of an RNA transcript.
  • the guide RNA comprises one or more mismatched nucleotides with the target RNA.
  • the guide RNA hybridizes to a target molecule that is diagnostic for a disease state, such as, but not limited to, cancer, or an immune disease.
  • the invention provides a ribonucleic acid (RNA) detection system, comprising a) a Type VI CRISPR-Cas effector protein capable of cleaving RNA, b) a guide RNA capable of binding to a target RNA, and c) an RNA-based cleavage inducible reporter capable of being non- specifically and detectably cleaved by the effector protein.
  • RNA detection system comprising a) a Type VI CRISPR-Cas effector protein capable of cleaving RNA, and b) an RNA-based cleavage inducible reporter capable of being non-specifically and detectably cleaved by the effector protein.
  • the RNA-based cleavage inducible reporter construct comprises a fluorochrome and a quencher.
  • the non-naturally occurring or engineered compositions, the vector systems, or the delivery systems as described in the present specification may be used for generating disease models and/or screening systems.
  • the non-naturally occurring or engineered compositions, the vector systems, or the delivery systems as described in the present specification may be used for: site-specific transcriptome editing or purturbation; nucleic acid sequence-specific interference; or multiplexed genome engineering.
  • the amount of gene product expressed may be greater than or less than the amount of gene product from a cell that does not have altered expression or edited genome.
  • the gene product may be altered in comparison with the gene product from a cell that does not have altered expression or edited genome.
  • FIG. 1 Comparison of Casl3a, Casl3b, and Casl3c orthologues
  • A HEK293 cells were transfected with different Cas 13 orthologues and Casl3 expression was characterized by fluorescence readout.
  • FIG. 2A-2B Transcript knockdown by Casl3 orthologs.
  • Various Casl3a, Casl3b, Casl3c orthologs were compared in as to knockdown of luciferase reporter gene expression in two experiments using two different guide nucleic acids, guide 1 (A) and guide 2 (B).
  • FIG. 3 Characterization of Cas 13 orthologs.
  • Various Cas 13 a, Cas 13b, and Casl3c orthologs were compared as to knockdown of a luciferase reporter gene by two different guide nucleic acids.
  • the Figure further highlights Casl3 orthologs that consistently knocked down gene expressin across different guide nucleic acids.
  • FIG. 4A-4B RNA binding by truncations of dCasl3b.
  • Various N-terminal and C- terminal truncations of dCasl3b are depicted.
  • RNA binding is incidated where there is ADAR- dependent RNA editing as measured by restoration of luciferase signal, comparing activity using targeting and non-targeting guides.
  • Amino acid positions correspond to amino acid positions of Prevotella sp. P5-125 Casl3b protein.
  • the figures herein are for illustrative purposes only and are not necessarily drawn to scale.
  • a "biological sample” may contain whole cells and/or live cells and/or cell debris.
  • the biological sample may contain (or be derived from) a "bodily fluid".
  • the present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof.
  • Biological samples include cell cultures, bodily fluids,
  • subject means a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • the term “crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components" of a Type V or Type VI CRISPR-Cas locus effector protein comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g., the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND Illumina, San Diego, CA
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • a guide sequence within a nucleic acid-targeting guide RNA
  • a guide sequence may direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence
  • the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid- targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein.
  • preferential targeting e.g., cleavage
  • cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence, and hence a nucleic acid-targeting guide may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence may be any RNA sequence.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (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 (IncRNA), and small cytoplasmatic RNA (scRNA).
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre- mRNA, and rRNA.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.
  • a nucleic acid-targeting guide is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal 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 example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence.
  • the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5') from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') from the guide sequence or spacer sequence.
  • the crRNA comprises a stem loop, preferably a single stem loop.
  • the direct repeat sequence forms a stem loop, preferably a single stem loop.
  • the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
  • the "tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize.
  • the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and crRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins. In a hairpin structure the portion of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop corresponds to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the sea sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the sea sequence or tracr sequence.
  • the degree of complementarity between the tracr sequence and sea sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the CRISPR-Cas, CRISPR-Casl3 or CRISPR system may be as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas") genes, including sequences encoding a Cas gene, in particular a Casl3 gene in the case of CRISPR-Casl3, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Casl3, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) e.g., RNA(s) to guide Casl3, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • 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).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • the section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell, and may include nucleic acids in or from mitochondrial, organelles, vesicles, liposomes or particles present within the cell. In some embodiments, especially for non-nuclear uses, NLSs are not preferred.
  • a CRISPR system comprises one or more nuclear exports signals (NESs).
  • NESs nuclear exports signals
  • a CRISPR system comprises one or more NLSs and one or more NESs.
  • direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
  • RNA capable of guiding Cas to a target genomic locus are used interchangeably as in foregoing cited documents such as WO 2014/093622 (PCT/US2013/074667).
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g.
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the guide sequence is 10 30 nucleotides long.
  • the ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide or RNA or sgRNA can be about or more than about 5, 10,
  • RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15,
  • an aspect of the invention is to reduce off-target interactions, e.g., reduce the guide interacting with a target sequence having low complementarity.
  • the invention involves mutations that result in the CRISPR-Cas system being able to distinguish between target and off- target sequences that have greater than 80% to about 95% complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (for instance, distinguishing between a target having 18 nucleotides from an off- target of 18 nucleotides having 1, 2 or 3 mismatches).
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%.
  • Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.
  • the guide RNA (capable of guiding Cas to atarget locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) may reside in a single RNA, i.e. an sgRNA (arranged in a 5 ' to 3 ' orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.
  • each RNA may be optimized to be shortened from their respective native lengths, and each may be independently chemically modified to protect from degradation by cellular RNase or otherwise increase stability.
  • the methods according to the invention as described herein comprehend inducing one or more mutations in a eukaryotic cell (in vitro, i.e. in an isolated eukaryotic cell) as herein discussed comprising delivering to cell a vector as herein discussed.
  • the mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 1, 5, 10, 1 1, 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 said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 5, 10, 1 1, 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 said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations include the introduction, deletion, or substitution of 10, 1 1, 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 said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the 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 said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the 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 said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • Cas mRNA and guide RNA For minimization of toxicity and off-target effect, it may be important to control the concentration of Cas mRNA and guide RNA delivered.
  • Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci.
  • Cas nickase mRNA for example S. pyogenes Cas9 with the D IOA mutation
  • Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667); or, via mutation as herein.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • a wild-type tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
  • guides of the invention comprise non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemically modifications.
  • Non-naturally occurring nucleic acids can include, for example, mixtures of naturally and non-naturally occurring nucleotides.
  • Non-naturally occurring nucleotides and/or nucleotide analogs may be modified at the ribose, phosphate, and/or base moiety.
  • a guide nucleic acid comprises ribonucleotides and non-ribonucleotides.
  • a guide comprises one or more ribonucleotides and one or more deoxyribonucleotides.
  • the guide comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, peptide nucleic acids (PNA), or bridged nucleic acids (BNA).
  • LNA locked nucleic acid
  • modified nucleotides include 2'-0-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs. Further examples of modified nucleotides include linkage of chemical moieties at the 2' position, including but not limited to peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG).
  • NLS nuclear localization sequence
  • PNA peptide nucleic acid
  • PEG polyethylene glycol
  • TEG tetraethyleneglycol
  • modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine ( ⁇ ), (me't), 5- methoxyuridine(5moU), inosine, 7-methylguanosine.
  • guide RNA chemical modifications include, without limitation, incorporation of 2'-0-methyl (M), 2 '-O-methyl-3 '-phosphorothioate (MS), phosphorothioate (PS), ⁇ -constrained ethyl(cEt), 2 ' -O-methyl-3 '-thioPACE (MSP), or 2 ' -O-methyl-3 '- phosphonoacetate (MP) at one or more terminal nucleotides.
  • Such chemically modified guides can comprise increased stability and increased activity as compared to unmodified guides, though on-target vs. off-target specificity is not predictable.
  • the 5' and/or 3' end of a guide RNA is modified by a variety of functional moieties including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags.
  • a guide comprises ribonucleotides in a region that binds to a target RNA and one or more deoxyribonucletides and/or nucleotide analogs in a region that binds to Cal3c.
  • deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide structures, such as, without limitation, 5' and/or 3' end, stem -loop regions, and the seed region.
  • the modification is not in the 5 '-handle of the stem-loop regions.
  • Chemical modification in the 5 '-handle of the stem-loop region of a guide may abolish its function (see Li, et al., Nature Biomedical Engineering, 2017, 1 :0066).
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 a guide is chemically modified.
  • 3-5 nucleotides at either the 3' or the 5 ' end of a guide is chemically modified.
  • only minor modifications are introduced in the seed region, such as 2'-F modifications.
  • 2'-F modification is introduced at the 3' end of a guide.
  • three to five nucleotides at the 5 ' and/or the 3 ' end of the guide are chemically modified with 2'-0-methyl (M), 2'-0-methyl-3'-phosphorothioate (MS), ⁇ -constrained ethyl(cEt), 2'-0- methyl-3'-thioPACE (MSP), or 2'-0-methyl-3'-phosphonoacetate (MP).
  • M 2'-0-methyl
  • MS 2'-0-methyl-3'-phosphorothioate
  • cEt ⁇ -constrained ethyl(cEt)
  • MSP 2'-0- methyl-3'-thioPACE
  • MP 2'-0-methyl-3'-phosphonoacetate
  • all of the phosphodiester bonds of a guide are substituted with phosphorothioates (PS) for enhancing levels of gene disruption.
  • PS phosphorothioates
  • more than five nucleotides at the 5' and/or the 3' end of the guide are chemically modified with 2'-0-Me, 2'-F or ⁇ -constrained ethyl (cEt).
  • cEt ⁇ -constrained ethyl
  • Such chemically modified guide can mediate enhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS, E7110-E7111).
  • a guide is modified to comprise a chemical moiety at its 3 ' and/or 5' end.
  • Such moieties include, but are not limited to amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), Rhodamine, peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG).
  • the chemical moiety is conjugated to the guide by a linker, such as an alkyl chain.
  • the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, protein, or nanoparticles.
  • Such chemically modified guide can be used to identify or enrich cells generically edited by a CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI: 10.7554).
  • 3 nucleotides at each ofthe 3' and 5' ends are chemically modified.
  • the modifications comprise 2'-0-methyl or phosphorothioate analogs.
  • 12 nucleotides in the tetraloop and 16 nucleotides in the stem -loop region are replaced with 2 '-O-methyl analogs.
  • Such chemical modifications improve in vivo editing and stability (see Finn et al., Cell Reports (2016), 22: 2227-2235).
  • more than 60 or 70 nucleotides of the guide are chemically modified.
  • this modification comprises replacement of nucleotides with 2 '-O-methyl or 2 '-fluoro nucleotide analogs or phosphorothioate (PS) modification of phosphodiester bonds.
  • the chemical modification comprises 2 '-O-methyl or 2 '-fluoro modification of guide nucleotides extending outside of the nuclease protein when the CRISPR complex is formed or PS modification of 20 to 30 or more nucleotides of the 3 '-terminus of the guide.
  • the chemical modification further comprises 2 '-O-methyl analogs at the 5' end of the guide or 2 '-fluoro analogs in the seed and tail regions.
  • Such chemical modifications improve stability to nuclease degradation and maintain or enhance genome- editing activity or efficiency, but modification of all nucleotides may abolish the function of the guide (see Yin et al., Nat. Biotech. (2016), 35(12): 1179-1187).
  • Such chemical modifications may be guided by knowledge of the structure of the CRISPR complex, including knowledge of the limited number of nuclease and RNA 2'-OH interactions (see Yin et al, Nat. Biotech. (2016), 35(12): 1179-1187).
  • one or more guide RNA nucleotides may be replaced with DNA nucleotides.
  • up to 2, 4, 6, 8, 10, or 12 RNA nucleotides of the 5'-end tail/seed guide region are replaced with DNA nucleotides.
  • the majority of guide RNA nucleotides at the 3' end are replaced with DNA nucleotides.
  • 16 guide RNA nucleotides at the 3' end are replaced with DNA nucleotides.
  • 8 guide RNA nucleotides of the 5 '-end tail/seed region and 16 RNA nucleotides at the 3 ' end are replaced with DNA nucleotides .
  • guide RNA nucleotides that extend outside of the nuclease protein when the CRISPR complex is formed are replaced with DNA nucleotides.
  • Such replacement of multiple RNA nucleotides with DNA nucleotides leads to decreased off-target activity but similar on-target activity compared to an unmodified guide; however, replacement of all RNA nucleotides at the 3 ' end may abolish the function of the guide (see Yin et al., Nat. Chem. Biol. (2016) 14, 311-316).
  • Such modifications may be guided by knowledge of the structure of the CRISPR complex, including knowledge of the limited number of nuclease and RNA 2' -OH interactions (see Yin et al., Nat. Chem. Biol. (2016) 14, 311-316).
  • the guide comprises a modified crRNA for Cpfl, having a 5'- handle and a guide segment further comprising a seed region and a 3 '-terminus.
  • the modified guide can be used with a Cpfl of any one of Acidaminococcus sp. BV3L6 Cpfl (AsCpfl); Francisella tularensis subsp. Novicida U112 Cpfl (FnCpfl); L.
  • bacterium MA2020 Cpfl Lb2Cpfl
  • Porphyromonas crevioricanis Cpfl PeCpfl
  • Porphyromonas macacae Cpfl PmCpfl
  • Candidatus Methanoplasma termitum Cpfl CtCpfl
  • Eubacterium eligens Cpfl EeCpfl
  • Moraxella bovoculi 237 Cpfl MbCpf 1
  • Prevotella disiens Cpfl PdCpfl
  • bacterium ND2006 Cpfl LbCpfl).
  • the modification to the guide is a chemical modification, an insertion, a deletion or a split.
  • the chemical modification includes, but is not limited to, incorporation of 2'-0-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine ( ⁇ ),
  • the guide comprises one or more of 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 some embodiments, all nucleotides are chemically modified.
  • one or more nucleotides in the seed region are chemically modified. In certain embodiments, one or more nucleotides in the 3 '-terminus are chemically modified. In certain embodiments, none of the nucleotides in the 5 '-handle is chemically modified. In some embodiments, the chemical modification in 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 in the 3 '-terminus are chemically modified.
  • nucleotides in the 3'-terminus improve gene cutting efficiency (see Li, et al., Nature Biomedical Engineering, 2017, 1 :0066).
  • 5 nucleotides in the 3'- terminus are replaced with 2'-fluoro analogues.
  • 10 nucleotides in the 3'-terminus are replaced with 2 '-fluoro analogues.
  • 5 nucleotides in the 3 '-terminus are replaced with 2'- O-methyl (M) analogs.
  • M O-methyl
  • 3 nucleotides at each of the 3' and 5' ends are chemically modified.
  • the modifications comprise 2'-0-methyl or phosphorothioate analogs.
  • 12 nucleotides in the tetraloop and 16 nucleotides in the stem -loop region are replaced with 2 '-O-methyl analogs.
  • Such chemical modifications improve in vivo editing and stability (see Finn et al., Cell Reports (2016), 22: 2227-2235).
  • the loop of the 5 '-handle of the guide is modified. In some embodiments, the loop of the 5 '-handle of the guide is modified to have a deletion, an insertion, a split, or chemical modifications. In certain embodiments, the loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence of UCUU, UUUU, UAUU, or UGUU. In some embodiments, the guide molecule forms a stemloop with a separate non-covalently linked sequence, which can be DNA or RNA.
  • the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-phosphodiester bond.
  • the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-nucleotide loop.
  • the tracr and tracr mate sequences are joined via a non-phosphodiester covalent linker.
  • covalent linker examples include but are not limited to a chemical moiety selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C-C bond forming groups such as Diels-Alder cyclo-addition pairs or ring -closing metathesis pairs, and Michael reaction pairs.
  • a chemical moiety selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates,
  • the tracr and tracr mate sequences are first synthesized using the standard phosphoramidite synthetic protocol (Herdewijn, P., ed., Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)).
  • the tracr or tracr mate sequences can be functionalized to contain an appropriate functional group for ligation using the standard protocol known in the art (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, thio semicarbazide, thiol, maleimide, haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide.
  • Examples of chemical bonds include, but are not limited to, those based on carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C-C bond forming groups such as Diels-Alder cyclo-addition pairs or ring- closing metathesis pairs, and Michael reaction pairs.
  • the tracr and tracr mate sequences can be chemically synthesized.
  • the chemical synthesis uses automated, solid-phase oligonucleotide synthesis machines with 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or 2'-thionocarbamate (2'-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989).
  • 2'-ACE 2'-acetoxyethyl orthoester
  • 2'-TC 2'-thionocarbamate
  • the tracr and tracr mate sequences can be covalently linked using various bioconjugation reactions, loops, bridges, and non-nucleotide links via modifications of sugar, internucleotide phosphodiester bonds, purine and pyrimidine residues.
  • the tracr and tracr mate sequences can be covalently linked using click chemistry. In some embodiments, the tracr and tracr mate sequences can be covalently linked using a triazole linker. In some embodiments, the tracr and tracr mate sequences can be covalently linked using Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and azide to yield a highly stable triazole linker (He et al., ChemBioChem (2015) 17: 1809-1812; WO 2016/186745).
  • the tracr and tracr mate sequences are covalently linked by ligating a 5'-hexyne tracrR A and a 3 '-azide crR A.
  • either or both of the 5'-hexyne tracrRNA and a 3 '-azide crRNA can be protected with 2'-acetoxyethl orthoester (2'-ACE) group, which can be subsequently removed using Dharmacon protocol (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18).
  • 2'-ACE 2'-acetoxyethl orthoester
  • the tracr and tracr mate sequences can be covalently linked via a linker (e.g., a non-nucleotide loop) that comprises a moiety such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye labeled RNAs, and non-naturally occurring nucleotide analogues.
  • a linker e.g., a non-nucleotide loop
  • a linker e.g., a non-nucleotide loop
  • a linker e.g., a non-nucleotide loop
  • a linker e.g., a non-nucleotide loop
  • suitable spacers for purposes of this invention include, but are not limited to, polyethers (e.g., polyethylene glycols, polyalcohols, polypropylene glycol or mixtures of efhylene and propylene glycols), polyamines group (e.g., spennine, spermidine and polymeric derivatives thereof), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof.
  • Suitable attachments include any moiety that can be added to the linker to add additional properties to the linker, such as but not limited to, fluorescent labels.
  • Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacyl glycerols and dialkyl glycerols, 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. The design of example linkers conjugating two RNA components are also described in WO 2004/015075.
  • the linker (e.g., a non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equivalent to about 0-16 nucleotides. In some embodiments, the linker has a length equivalent to about 0-8 nucleotides. In some embodiments, the linker has a length equivalent to about 0-4 nucleotides. In some embodiments, the linker has a length equivalent to about 2 nucleotides.
  • Example linker design is also described in WO2011/008730.
  • a typical Type II Cas sgRNA comprises (in 5' to 3' direction): a guide sequence, a poly U tract, a first complimentary stretch (the "repeat"), a loop (tetraloop), a second complimentary stretch (the "anti-repeat” being complimentary to the repeat), a stem, and further stem loops and stems and a poly A (often poly U in RNA) tail (terminator).
  • a guide sequence a poly U tract
  • a first complimentary stretch the "repeat”
  • a loop traloop
  • the anti-repeat being complimentary to the repeat
  • stem and further stem loops and stems and a poly A (often poly U in RNA) tail (terminator).
  • certain aspects of guide architecture are retained, certain aspect of guide architecture cam be modified, for example by addition, subtraction, or substitution of features, whereas certain other aspects of guide architecture are maintained.
  • Preferred locations for engineered sgRNA modifications include guide termini and regions of the sgRNA that are exposed when complexed with CRISPR protein and/or target, for example the tetraloop and/or loop2.
  • guides of the invention comprise specific binding sites (e.g. aptamers) for adapter proteins, which may comprise one or more functional domains (e.g. via fusion protein).
  • CRISPR complex i.e. CRISPR enzyme binding to guide and target
  • the adapter proteins bind and, the functional domain associated with the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective.
  • the functional domain is a transcription activator (e.g. VP64 or p65)
  • the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target.
  • a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g. Fokl) will be advantageously positioned to cleave or partially cleave the target.
  • the one or more modified guide may be modified at the tetra loop, the stem loop 1, stem loop 2, or stem loop 3, as described herein, preferably at either the tetra loop or stem loop 2, and most preferably at both the tetra loop and stem loop 2.
  • the repeat: anti repeat duplex will be apparent from the secondary structure of the sgRNA. It may be typically a first complimentary stretch after (in 5 ' to 3 ' direction) the poly U tract and before the tetraloop; and a second complimentary stretch after (in 5' to 3' direction) the tetraloop and before the poly A tract.
  • the first complimentary stretch (the "repeat") is complimentary to the second complimentary stretch (the "anti -repeat").
  • the anti-repeat sequence is the complimentary sequence of the repeat and in terms to A-U or C-G base pairing, but also in terms of the fact that the anti -repeat is in the reverse orientation due to the tetraloop.
  • modification of guide architecture comprises replacing bases in stemloop 2.
  • “actt” (“acuu” in RNA) and “aagt” (“aagu” in RNA) bases in stemloop2 are replaced with “cgcc” and “gcgg”.
  • “actt” and “aagt” bases in stemloop2 are replaced with complimentary GC-rich regions of 4 nucleotides.
  • the complimentary GC-rich regions of 4 nucleotides are "cgcc” and "gcgg” (both in 5 ' to 3 ' direction).
  • the complimentary GC-rich regions of 4 nucleotides are "gcgg” and “cgcc” (both in 5' to 3' direction).
  • Other combination of C and G in the complimentary GC-rich regions of 4 nucleotides will be apparent including CCCC and GGGG.
  • the stemloop 2 e.g., "ACTTgtttAAGT” can be replaced by any "XXXXgtttYYYY", e.g., where XXXX and YYYY represent any complementary sets of nucleotides that together will base pair to each other to create a stem.
  • the stem comprises at least about 4bp comprising complementary X and Y sequences, although stems of more, e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs are also contemplated.
  • stems of more, e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs are also contemplated.
  • X2-12 and Y2-12 (wherein X and Y represent any complementary set of nucleotides) may be contemplated.
  • the stem made of the X and Y nucleotides, together with the "gttt,” will form a complete hairpin in the overall secondary structure; and, this may be advantageous and the amount of base pairs can be any amount that forms a complete hairpin.
  • any complementary X:Y basepairing sequence (e.g., as to length) is tolerated, so long as the secondary structure of the entire sgR A is preserved.
  • the stem can be a form of X:Y basepairing that does not disrupt the secondary structure of the whole sgRNA in that it has a DR:tracr duplex, and 3 stemloops.
  • the "gttt" tetraloop that connects ACTT and AAGT (or any alternative stem made of X:Y basepairs) can be any sequence of the same length (e.g., 4 basepair) or longer that does not interrupt the overall secondary structure of the sgRNA.
  • the stemloop can be something that further lengthens stemloop2, e.g. can be MS2 aptamer.
  • the stemloop3 "GGCACCGagtCGGTGC” can likewise take on a "XXXXXXXagtYYYYYYY” form, e.g., wherein X7 and Y7 represent any complementary sets of nucleotides that together will base pair to each other to create a stem.
  • the stem comprises about 7bp comprising complementary X and Y sequences, although stems of more or fewer basepairs are also contemplated.
  • the stem made of the X and Y nucleotides, together with the "agt”, will form a complete hairpin in the overall secondary structure.
  • any complementary X:Y basepairing sequence is tolerated, so long as the secondary structure of the entire sgRNA is preserved.
  • the stem can be a form of X:Y basepairing that doesn't disrupt the secondary structure of the whole sgRNA in that it has a DR:tracr duplex, and 3 stemloops.
  • the "agt" sequence of the stemloop 3 can be extended or be replaced by an aptamer, e.g., a MS2 aptamer or sequence that otherwise generally preserves the architecture of stemloop3.
  • each X and Y pair can refer to any basepair.
  • non-Watson Crick basepairing is contemplated, where such pairing otherwise generally preserves the architecture of the stemloop at that position.
  • the DR:tracrR A duplex can be replaced with the form: gYYYYag(N)N NxxxxN (AAN)uuRRRRu (using standard IUPAC nomenclature for nucleotides), wherein (N) and (AAN) represent part of the bulge in the duplex, and "xxxx" represents a linker sequence.
  • NNNN on the direct repeat can be anything so long as it basepairs with the corresponding NNNN portion of the tracrRNA.
  • the DR:tracrRNA duplex can be connected by a linker of any length (xxxx%), any base composition, as long as it doesn't alter the overall structure.
  • the sgRNA structural requirement is to have a duplex and 3 stemloops.
  • the actual sequence requirement for many of the particular base requirements are lax, in that the architecture of the DR:tracrRNA duplex should be preserved, but the sequence that creates the architecture, i.e., the stems, loops, bulges, etc., may be alterred.
  • One guide with a first aptamer/RNA-binding protein pair can be linked or fused to an activator, whilst a second guide with a second aptamer/RNA-binding protein pair can be linked or fused to a repressor.
  • the guides are for different targets (loci), so this allows one gene to be activated and one repressed. For example, the following schematic shows such an approach:
  • the present invention also relates to orthogonal PP7/MS2 gene targeting.
  • sgRNA targeting different loci are modified with distinct RNA loops in order to recruit MS2-VP64 or PP7- SID4X, which activate and repress their target loci, respectively.
  • PP7 is the RNA-binding coat protein of the bacteriophage Pseudomonas. Like MS2, it binds a specific RNA sequence and secondary structure.
  • the PP7 RNA-recognition motif is distinct from that of MS2. Consequently, PP7 and MS2 can be multiplexed to mediate distinct effects at different genomic loci simultaneously.
  • an sgRNA targeting locus A can be modified with MS2 loops, recruiting MS2-VP64 activators, while another sgRNA targeting locus B can be modified with PP7 loops, recruiting PP7-SID4X repressor domains.
  • dCasl3 can thus mediate orthogonal, locus-specific modifications. This principle can be extended to incorporate other orthogonal RNA-binding proteins such as Q-beta.
  • An alternative option for orthogonal repression includes incorporating non-coding RNA loops with transactive repressive function into the guide (either at similar positions to the MS2/PP7 loops integrated into the guide or at the 3' terminus of the guide).
  • guides were designed with non- coding (but known to be repressive) RNA loops (e.g. using the Alu repressor (in R A) that interferes with R A polymerase II in mammalian cells).
  • the Alu RNA sequence was located: in place of the MS2 RNA sequences as used herein (e.g. at tetraloop and/or stem loop 2); and/or at 3' terminus of the guide. This gives possible combinations of MS2, PP7 or Alu at the tetraloop and/or stemloop 2 positions, as well as, optionally, addition of Alu at the 3' end of the guide (with or without a linker).
  • the adaptor protein may be associated (preferably linked or fused to) one or more activators or one or more repressors.
  • the adaptor protein may be associated with a first activator and a second activator.
  • the first and second activators may be the same, but they are preferably different activators.
  • Three or more or even four or more activators (or repressors) may be used, but package size may limit the number being higher than 5 different functional domains.
  • Linkers are preferably used, over a direct fusion to the adaptor protein, where two or more functional domains are associated with the adaptor protein. Suitable linkers might include the GlySer linker.
  • the enzyme-guide complex as a whole may be associated with two or more functional domains.
  • there may be two or more functional domains associated with the enzyme or there may be two or more functional domains associated with the guide (via one or more adaptor proteins), or there may be one or more functional domains associated with the enzyme and one or more functional domains associated with the guide (via one or more adaptor proteins).
  • the fusion between the adaptor protein and the activator or repressor may include a linker.
  • a linker For example, GlySer linkers GGGS can be used. They can be used in repeats of 3 ((GGGGS)3) or 6, 9 or even 12 or more, to provide suitable lengths, as required.
  • Linkers can be used between the RNA-binding protein and the functional domain (activator or repressor), or between the CRISPR Enzyme (Casl3) and the functional domain (activator or repressor). The linkers the user to engineer appropriate amounts of "mechanical flexibility".
  • Guide RNAs comprising a dead guide sequence may be used in the present invention [00130]
  • the invention provides guide sequences which are modified in a manner which allows for formation of the CRISPR complex and successful binding to the target, while at the same time, not allowing for successful nuclease activity (i.e. without nuclease activity / without indel activity).
  • modified guide sequences are referred to as "dead guides” or “dead guide sequences”.
  • These dead guides or dead guide sequences can be thought of as catalytically inactive or conformationally inactive with regard to nuclease activity.
  • Nuclease activity may be measured using surveyor analysis or deep sequencing as commonly used in the art, preferably surveyor analysis. Similarly, dead guide sequences may not sufficiently engage in productive base pairing with respect to the ability to promote catalytic activity or to distinguish on-target and off-target binding activity.
  • the surveyor assay involves purifying and amplifying a CRISPR target site for a gene and forming heteroduplexes with primers amplifying the CRISPR target site. After re-anneal, the products are treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) following the manufacturer's recommended protocols, analyzed on gels, and quantified based upon relative band intensities.
  • the invention provides a non-naturally occurring or engineered composition Casl3 CRISPR-Cas system comprising a functional Casl3 as described herein, and guide RNA (gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the Casl3 CRISPR-Cas system is directed to a genomic locus of interest in a cell without detectable indel activity resultant from nuclease activity of a non-mutant Casl3 enzyme of the system as detected by a SURVEYOR assay.
  • gRNA guide RNA
  • a gRNA comprising a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the Cas 13 CRISPR-Cas system is directed to a genomic locus of interest in a cell without detectable indel activity resultant from nuclease activity of a non-mutant Cas 13 enzyme of the system as detected by a SURVEYOR assay is herein termed a "dead gRNA".
  • a dead gRNA any of the gRNAs according to the invention as described herein elsewhere may be used as dead gRNAs / gRNAs comprising a dead guide sequence as described herein below. Any of the methods, products, compositions and uses as described herein elsewhere is equally applicable with the dead gRNAs / gRNAs comprising a dead guide sequence as further detailed below.
  • the ability of a dead guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the dead guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the dead guide sequence to be tested and a control guide sequence different from the test dead guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a dead guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Dead guide sequences are shorter than respective guide sequences which result in active Cas 13 -specific indel formation.
  • Dead guides are 5%, 10%, 20%, 30%, 40%, 50%, shorter than respective guides directed to the same Cas 13 leading to active Cas 13 -specific indel formation.
  • one aspect of gRNA - Casl3 specificity is the direct repeat sequence, which is to be appropriately linked to such guides.
  • structural data available for validated dead guide sequences may be used for designing Cas 13 specific equivalents.
  • Structural similarity between, e.g., the orthologous nuclease domains RuvC of two or more Cas 13 effector proteins may be used to transfer design equivalent dead guides.
  • the dead guide herein may be appropriately modified in length and sequence to reflect such Cas 13 specific equivalents, allowing for formation of the CRISPR complex and successful binding to the target, while at the same time, not allowing for successful nuclease activity.
  • dead guides in the context herein as well as the state of the art provides a surprising and unexpected platform for network biology and/or systems biology in both in vitro, ex vivo, and in vivo applications, allowing for multiplex gene targeting, and in particular bidirectional multiplex gene targeting.
  • addressing multiple targets for example for activation, repression and/or silencing of gene activity, has been challenging and in some cases not possible.
  • multiple targets, and thus multiple activities may be addressed, for example, in the same cell, in the same animal, or in the same patient. Such multiplexing may occur at the same time or staggered for a desired timeframe.
  • the dead guides now allow for the first time to use gRNA as a means for gene targeting, without the consequence of nuclease activity, while at the same time providing directed means for activation or repression.
  • Guide RNA comprising a dead guide may be modified to further include elements in a manner which allow for activation or repression of gene activity, in particular protein adaptors (e.g. aptamers) as described herein elsewhere allowing for functional placement of gene effectors (e.g. activators or repressors of gene activity).
  • protein adaptors e.g. aptamers
  • gene effectors e.g. activators or repressors of gene activity.
  • One example is the incorporation of aptamers, as explained herein and in the state of the art.
  • gRNA By engineering the gRNA comprising a dead guide to incorporate protein- interacting aptamers (Konermann et al, "Genome-scale transcription activation by an engineered CRISPR- Casl3 complex," doi: 10.1038/naturel4136, inco ⁇ orated herein by reference), one may assemble a synthetic transcription activation complex consisting of multiple distinct effector domains. Such may be modeled after natural transcription activation processes. For example, an aptamer, which selectively binds an effector (e.g. an activator or repressor; dimerized MS2 bacteriophage coat proteins as fusion proteins with an activator or repressor), or a protein which itself binds an effector (e.g.
  • an effector e.g. an activator or repressor; dimerized MS2 bacteriophage coat proteins as fusion proteins with an activator or repressor
  • a protein which itself binds an effector e.g.
  • the fusion protein MS2- VP64 binds to the tetraloop and/or stem-loop 2 and in turn mediates transcriptional up-regulation, for example iorNeurog2.
  • Other transcriptional activators are, for example, VP64. P65, HSF1, and MyoD l.
  • a gRNA of the invention which comprises a dead guide, wherein the gRNA further comprises modifications which provide for gene activation or repression, as described herein.
  • the dead gRNA may comprise one or more aptamers.
  • the aptamers may be specific to gene effectors, gene activators or gene repressors.
  • the aptamers may be specific to a protein which in turn is specific to and recruits / binds a specific gene effector, gene activator or gene repressor. If there are multiple sites for activator or repressor recruitment, it is preferred that the sites are specific to either activators or repressors.
  • the sites may be specific to the same activators or same repressors.
  • the sites may also be specific to different activators or different repressors.
  • the gene effectors, gene activators, gene repressors may be present in the form of fusion proteins.
  • the dead gRNA as described herein or the Casl3 CRISPR-Cas complex as described herein includes a non-naturally occurring or engineered composition comprising two or more adaptor proteins, wherein each protein is associated with one or more functional domains and wherein the adaptor protein binds to the distinct RNA sequence(s) inserted into the at least one loop of the dead gRNA.
  • an aspect provides a non-naturally occurring or engineered composition
  • a guide RNA comprising a dead guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell
  • the dead guide sequence is as defined herein
  • a Cas 13 comprising at least one or more nuclear localization sequences, wherein the Cas 13 optionally comprises at least one mutation wherein at least one loop of the dead gRNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or, wherein the dead gRNA is modified to have at least one non-coding functional loop, and wherein the composition comprises two or more adaptor proteins, wherein the each protein is associated with one or more functional domains.
  • the adaptor protein is a fusion protein comprising the functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain,
  • the at least one loop of the dead gR A is not modified by the insertion of distinct RNA sequence(s) that bind to the two or more adaptor proteins.
  • the one or more functional domains associated with the adaptor protein is a transcriptional activation domain.
  • the one or more functional domains associated with the adaptor protein is a transcriptional activation domain comprising VP64, p65, MyoDl, HSF1, RTA or SET7/9.
  • the one or more functional domains associated with the adaptor protein is a transcriptional repressor domain.
  • the transcriptional repressor domain is a KRAB domain.
  • the transcriptional repressor domain is a NuE domain, NcoR domain,
  • SID domain or a SID4X domain.
  • At least one of the one or more functional domains associated with the adaptor protein have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity.
  • the DNA cleavage activity is due to a Fokl nuclease.
  • the dead gRNA is modified so that, after dead gRNA binds the adaptor protein and further binds to the Casl3 and target, the functional domain is in a spatial orientation allowing for the functional domain to function in its attributed function.
  • the at least one loop of the dead gRNA is tetra loop and/or loop2.
  • the tetra loop and loop 2 of the dead gRNA are modified by the insertion of the distinct RNA sequence(s).
  • the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins is an aptamer sequence.
  • the aptamer sequence is two or more aptamer sequences specific to the same adaptor protein.
  • the aptamer sequence is two or more aptamer sequences specific to different adaptor protein.
  • the adaptor protein comprises MS2, PP7, Q , F2, GA, fir, JP501, Ml 2, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, (
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell, optionally a mouse cell.
  • the mammalian cell is a human cell.
  • a first adaptor protein is associated with a p65 domain and a second adaptor protein is associated with a HSF1 domain.
  • the composition comprises a Casl3 CRISPR-Cas complex having at least three functional domains, at least one of which is associated with the Casl3 and at least two of which are associated with dead gRNA.
  • the composition further comprises a second gRNA, wherein the second gRNA is a live gRNA capable of hybridizing to a second target sequence such that a second Casl3 CRISPR-Cas system is directed to a second genomic locus of interest in a cell with detectable indel activity at the second genomic locus resultant from nuclease activity of the Casl3 enzyme of the system.
  • the second gRNA is a live gRNA capable of hybridizing to a second target sequence such that a second Casl3 CRISPR-Cas system is directed to a second genomic locus of interest in a cell with detectable indel activity at the second genomic locus resultant from nuclease activity of the Casl3 enzyme of the system.
  • the composition further comprises a plurality of dead gRNAs and/or a plurality of live gRNAs.
  • One aspect of the invention is to take advantage of the modularity and customizability of the gRNA scaffold to establish a series of gRNA scaffolds with different binding sites (in particular aptamers) for recruiting distinct types of effectors in an orthogonal manner.
  • replacement of the MS2 stem-loops with PP7-interacting stem-loops may be used to bind / recruit repressive elements, enabling multiplexed bidirectional transcriptional control.
  • gRNA comprising a dead guide may be employed to provide for multiplex transcriptional control and preferred bidirectional transcriptional control. This transcriptional control is most preferred of genes.
  • one or more gRNA comprising dead guide(s) may be employed in targeting the activation of one or more target genes.
  • one or more gRNA comprising dead guide(s) may be employed in targeting the repression of one or more target genes.
  • Such a sequence may be applied in a variety of different combinations, for example the target genes are first repressed and then at an appropriate period other targets are activated, or select genes are repressed at the same time as select genes are activated, followed by further activation and/or repression.
  • multiple components of one or more biological systems may advantageously be addressed together.
  • the invention provides nucleic acid molecule(s) encoding dead gRNA or the Casl3 CRISPR-Cas complex or the composition as described herein.
  • the invention provides a vector system comprising: a nucleic acid molecule encoding dead guide RNA as defined herein.
  • the vector system further comprises a nucleic acid molecule(s) encoding Casl3.
  • the vector system further comprises a nucleic acid molecule(s) encoding (live) gRNA.
  • the nucleic acid molecule or the vector further comprises regulatory element(s) operable in a eukaryotic cell operably linked to the nucleic acid molecule encoding the guide sequence (gRNA) and/or the nucleic acid molecule encoding Cas9 and/or the optional nuclear localization sequence(s).
  • structural analysis may also be used to study interactions between the dead guide and the active Cas l3 nuclease that enable DNA binding, but no DNA cutting.
  • amino acids important for nuclease activity of Cas l3 are determined. Modification of such amino acids allows for improved Cas l3 enzymes used for gene editing.
  • a further aspect is combining the use of dead guides as explained herein with other applications of CRISPR, as explained herein as well as known in the art.
  • gRNA comprising dead guide(s) for targeted multiplex gene activation or repression or targeted multiplex bidirectional gene activation / repression may be combined with gRNA comprising guides which maintain nuclease activity, as explained herein.
  • Such gRNA comprising guides which maintain nuclease activity may or may not further include modifications which allow for repression of gene activity (e.g. aptamers).
  • Such gRNA comprising guides which maintain nuclease activity may or may not further include modifications which allow for activation of gene activity (e.g. aptamers).
  • multiplex gene control e.g. multiplex gene targeted activation without nuclease activity / without indel activity may be provided at the same time or in combination with gene targeted repression with nuclease activity).
  • gRNA e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5 comprising dead guide(s) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene activators; 2) may be combined with one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) comprising dead guide(s) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene repressors. 1) and/or 2) may then be combined with 3) one or more gRNA (e.g.
  • This combination can then be carried out in turn with 1) + 2) + 3) with 4) one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene activators.
  • This combination can then be carried in turn with 1) + 2) + 3) + 4) with 5) one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene repressors.
  • the invention provides an algorithm for designing, evaluating, or selecting a dead guide RNA targeting sequence (dead guide sequence) for guiding a Casl3 CRISPR-Cas system to a target gene locus.
  • dead guide RNA specificity relates to and can be optimized by varying i) GC content and ii) targeting sequence length.
  • the invention provides an algorithm for designing or evaluating a dead guide RNA targeting sequence that minimizes off-target binding or interaction of the dead guide RNA.
  • the algorithm for selecting a dead guide RNA targeting sequence for directing a CRISPR system to a gene locus in an organism comprises a) locating one or more CRISPR motifs in the gene locus, analyzing the 20 nt sequence downstream of each CRISPR motif by i) determining the GC content of the sequence; and ii) determining whether there are off-target matches of the 15 downstream nucleotides nearest to the CRISPR motif in the genome of the organism, and c) selecting the 15 nucleotide sequence for use in a dead guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified.
  • the sequence is selected for a targeting sequence if the GC content is 60% or less.
  • the sequence is selected for a targeting sequence if the GC content is 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less. In an embodiment, two or more sequences of the gene locus are analyzed and the sequence having the lowest GC content, or the next lowest GC content, or the next lowest GC content is selected. In an embodiment, the sequence is selected for a targeting sequence if no off-target matches are identified in the genome of the organism. In an embodiment, the targeting sequence is selected if no off-target matches are identified in regulatory sequences of the genome.
  • the invention provides a method of selecting a dead guide RNA targeting sequence for directing a functionalized CRISPR system to a gene locus in an organism, which comprises: a) locating one or more CRISPR motifs in the gene locus; b) analyzing the 20 nt sequence downstream of each CRISPR motif by: i) determining the GC content of the sequence; and ii) determining whether there are off-target matches of the first 15 nt of the sequence in the genome of the organism; c) selecting the sequence for use in a guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In an embodiment, the sequence is selected if the GC content is 50% or less.
  • the sequence is selected if the GC content is 40% or less. In an embodiment, the sequence is selected if the GC content is 30% or less. In an embodiment, two or more sequences are analyzed and the sequence having the lowest GC content is selected. In an embodiment, off-target matches are determined in regulatory sequences of the organism. In an embodiment, the gene locus is a regulatory region. An aspect provides a dead guide RNA comprising the targeting sequence selected according to the aforementioned methods.
  • the invention provides a dead guide RNA for targeting a functionalized CRISPR system to a gene locus in an organism.
  • the dead guide RNA comprises a targeting sequence wherein the CG content of the target sequence is 70% or less, and the first 15 nt of the targeting sequence does not match an off-target sequence downstream from a CRISPR motif in the regulatory sequence of another gene locus in the organism.
  • the GC content of the targeting sequence 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less.
  • the GC content of the targeting sequence is from 70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%.
  • the targeting sequence has the lowest CG content among potential targeting sequences of the locus.
  • the first 15 nt of the dead guide match the target sequence.
  • first 14 nt of the dead guide match the target sequence.
  • the first 13 nt of the dead guide match the target sequence.
  • first 12 nt of the dead guide match the target sequence.
  • first 11 nt of the dead guide match the target sequence.
  • the first 10 nt of the dead guide match the target sequence.
  • the first 15 nt of the dead guide does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus.
  • the first 14 nt, or the first 13 nt of the dead guide, or the first 12 nt of the guide, or the first 11 nt of the dead guide, or the first 10 nt of the dead guide does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus.
  • the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt of the dead guide do not match an off-target sequence downstream from a CRISPR motif in the genome.
  • the dead guide RNA includes additional nucleotides at the 3 '-end that do not match the target sequence.
  • a dead guide RNA that includes the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt downstream of a CRISPR motif can be extended in length at the 3 ' end to 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, or longer.
  • the invention provides a method for directing a Casl3 CRISPR-Cas system, including but not limited to a dead Casl3 (dCasl3) or functionalized Casl3 system (which may comprise a functionalized Casl3 or functionalized guide) to a gene locus.
  • the invention provides a method for selecting a dead guide RNA targeting sequence and effecting gene regulation of a target gene locus by a functionalized Casl3 CRISPR-Cas system.
  • the method is used to effect target gene regulation while minimizing off-target effects.
  • the invention provides a method for selecting two or more dead guide RNA targeting sequences and effecting gene regulation of two or more target gene loci by a functionalized Casl3 CRISPR-Cas system.
  • the method is used to effect regulation of two or more target gene loci while minimizing off-target effects.
  • the invention provides a method of selecting a dead guide RNA targeting sequence for directing a functionalized Casl3 to a gene locus in an organism, which comprises: a) locating one or more CRISPR motifs in the gene locus; b) analyzing the sequence downstream of each CRISPR motif by: i) selecting 10 to 15 nt adjacent to the CRISPR motif, ii) determining the GC content of the sequence; and c) selecting the 10 to 15 nt sequence as a targeting sequence for use in a guide RNA if the GC content of the sequence is 40% or more. In an embodiment, the sequence is selected if the GC content is 50% or more.
  • the sequence is selected if the GC content is 60% or more. In an embodiment, the sequence is selected if the GC content is 70% or more. In an embodiment, two or more sequences are analyzed and the sequence having the highest GC content is selected. In an embodiment, the method further comprises adding nucleotides to the 3' end of the selected sequence which do not match the sequence downstream of the CRISPR motif.
  • An aspect provides a dead guide RNA comprising the targeting sequence selected according to the aforementioned methods.
  • the invention provides a dead guide RNA for directing a functionalized CRISPR system to a gene locus in an organism wherein the targeting sequence of the dead guide RNA consists of 10 to 15 nucleotides adjacent to the CRISPR motif of the gene locus, wherein the CG content of the target sequence is 50% or more.
  • the dead guide RNA further comprises nucleotides added to the 3 ' end of the targeting sequence which do not match the sequence downstream of the CRISPR motif of the gene locus.
  • the invention provides for a single effector to be directed to one or more, or two or more gene loci.
  • the effector is associated with a Casl3, and one or more, or two or more selected dead guide RNAs are used to direct the Cas 13 -associated effector to one or more, or two or more selected target gene loci.
  • the effector is associated with one or more, or two or more selected dead guide RNAs, each selected dead guide RNA, when complexed with a Cas 13 enzyme, causing its associated effector to localize to the dead guide RNA target.
  • CRISPR systems modulates activity of one or more, or two or more gene loci subject to regulation by the same transcription factor.
  • the invention provides for two or more effectors to be directed to one or more gene loci.
  • two or more dead guide RNAs are employed, each of the two or more effectors being associated with a selected dead guide RNA, with each of the two or more effectors being localized to the selected target of its dead guide RNA.
  • CRISPR systems modulates activity of one or more, or two or more gene loci subject to regulation by different transcription factors.
  • two or more transcription factors are localized to different regulatory sequences of a single gene.
  • two or more transcription factors are localized to different regulatory sequences of different genes.
  • one transcription factor is an activator.
  • one transcription factor is an inhibitor. In certain embodiments, one transcription factor is an activator and another transcription factor is an inhibitor. In certain embodiments, gene loci expressing different components of the same regulatory pathway are regulated. In certain embodiments, gene loci expressing components of different regulatory pathways are regulated.
  • the invention also provides a method and algorithm for designing and selecting dead guide RNAs that are specific for target DNA cleavage or target binding and gene regulation mediated by an active Casl3 CRISPR-Cas system.
  • the Casl3 CRISPR-Cas system provides orthogonal gene control using an active Casl3 which cleaves target DNA at one gene locus while at the same time binds to and promotes regulation of another gene locus.
  • the invention provides an method of selecting a dead guide RNA targeting sequence for directing a functionalized Casl3 to a gene locus in an organism, without cleavage, which comprises a) locating one or more CRISPR motifs in the gene locus; b) analyzing the sequence downstream of each CRISPR motif by i) selecting 10 to 15 nt adjacent to the CRISPR motif, ii) determining the GC content of the sequence, and c) selecting the 10 to 15 nt sequence as a targeting sequence for use in a dead guide RNA if the GC content of the sequence is 30% more, 40% or more.
  • the GC content of the targeting sequence is 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. In certain embodiments, the GC content of the targeting sequence is from 30% to 40% or from 40% to 50% or from 50% to 60% or from 60% to 70%. In an embodiment of the invention, two or more sequences in a gene locus are analyzed and the sequence having the highest GC content is selected.
  • the portion of the targeting sequence in which GC content is evaluated is 10 to 15 contiguous nucleotides of the 15 target nucleotides nearest to the PAM.
  • the portion of the guide in which GC content is considered is the 10 to 11 nucleotides or 11 to 12 nucleotides or 12 to 13 nucleotides or 13, or 14, or 15 contiguous nucleotides of the 15 nucleotides nearest to the PAM.
  • the invention further provides an algorithm for identifying dead guide RNAs which promote CRISPR system gene locus cleavage while avoiding functional activation or inhibition. It is observed that increased GC content in dead guide RNAs of 16 to 20 nucleotides coincides with increased DNA cleavage and reduced functional activation.
  • efficiency of functionalized Casl3 can be increased by addition of nucleotides to the 3 ' end of a guide RNA which do not match a target sequence downstream of the CRISPR motif.
  • a guide RNA which do not match a target sequence downstream of the CRISPR motif.
  • shorter guides may be less likely to promote target cleavage, but are also less efficient at promoting CRISPR system binding and functional control.
  • addition of nucleotides that don't match the target sequence to the 3' end of the dead guide RNA increase activation efficiency while not increasing undesired target cleavage.
  • the invention also provides a method and algorithm for identifying improved dead guide RNAs that effectively promote CRISPRP system function in DNA binding and gene regulation while not promoting DNA cleavage.
  • the invention provides a dead guide RNA that includes the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 1 1 nt downstream of a CRISPR motif and is extended in length at the 3' end by nucleotides that mismatch the target to 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, or longer.
  • the invention provides a method for effecting selective orthogonal gene control.
  • dead guide selection according to the invention, taking into account guide length and GC content, provides effective and selective transcription control by a functional Casl3 CRISPR-Cas system, for example to regulate transcription of a gene locus by activation or inhibition and minimize off-target effects.
  • the invention also provides effective orthogonal regulation of two or more target loci.
  • orthogonal gene control is by activation or inhibition of two or more target loci. In certain embodiments, orthogonal gene control is by activation or inhibition of one or more target locus and cleavage of one or more target locus.
  • the invention provides a cell comprising a non-naturally occurring Casl3 CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein wherein the expression of one or more gene products has been altered.
  • the expression in the cell of two or more gene products has been altered.
  • the invention also provides a cell line from such a cell.
  • the invention provides a multicellular organism comprising one or more cells comprising a non-naturally occurring Casl3 CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein.
  • the invention provides a product from a cell, cell line, or multicellular organism comprising a non-naturally occurring Casl3 CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein.
  • a further aspect of this invention is the use of gRNA comprising dead guide(s) as described herein, optionally in combination with gRNA comprising guide(s) as described herein or in the state of the art, in combination with systems e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) which are engineered for either overexpression of Casl3 or preferably knock in Casl3.
  • systems e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice
  • one or more dead gRNAs may be provided to direct multiplex gene regulation, and preferably multiplex bidirectional gene regulation.
  • the one or more dead gRNAs may be provided in a spatially and temporally appropriate manner if necessary or desired (for example tissue specific induction of Casl3 expression).
  • tissue specific induction of Casl3 expression for example tissue specific induction of Casl3 expression.
  • both gRNAs comprising dead guides or gRNAs comprising guides are equally effective.
  • a further aspect of this invention is the use of gRNA comprising dead guide(s) as described herein, optionally in combination with gRNA comprising guide(s) as described herein or in the state of the art, in combination with systems (e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) which are engineered for knockout Casl3 CRISPR-Cas.
  • systems e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice
  • the combination of dead guides as described herein with CRISPR applications described herein and CRISPR applications known in the art results in a highly efficient and accurate means for multiplex screening of systems (e.g. network biology).
  • Such screening allows, for example, identification of specific combinations of gene activities for identifying genes responsible for diseases (e.g. on/off combinations), in particular gene related diseases.
  • a preferred application of such screening is cancer.
  • screening for treatment for such diseases is included in the invention.
  • Cells or animals may be exposed to aberrant conditions resulting in disease or disease like effects.
  • Candidate compositions may be provided and screened for an effect in the desired multiplex environment. For example a patient's cancer cells may be screened for which gene combinations will cause them to die, and then use this information to establish appropriate therapies.
  • the invention provides a kit comprising one or more of the components described herein.
  • the kit may include dead guides as described herein with or without guides as described herein.
  • the structural information provided herein allows for interrogation of dead gRNA interaction with the target DNA and the Casl3 permitting engineering or alteration of dead gRNA structure to optimize functionality of the entire Casl3 CRISPR-Cas system.
  • loops of the dead gRNA may be extended, without colliding with the Casl3 protein by the insertion of adaptor proteins that can bind to RNA.
  • adaptor proteins can further recruit effector proteins or fusions which comprise one or more functional domains.
  • the functional domain is a transcriptional activation domain, preferably VP64.
  • the functional domain is a transcription repression domain, preferably KRAB.
  • the transcription repression domain is SID, or concatemers of SID (e.g. SID4X).
  • the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided.
  • the functional domain is an activation domain, which may be the P65 activation domain.
  • An aspect of the invention is that the above elements are comprised in a single composition or comprised in individual compositions. These compositions may advantageously be applied to a host to elicit a functional effect on the genomic level.
  • the dead gRNA are modified in a manner that provides specific binding sites (e.g. aptamers) for adapter proteins comprising one or more functional domains (e.g. via fusion protein) to bind to.
  • the modified dead gRNA are modified such that once the dead gRNA forms a CRISPR complex (i.e. Cas 13 binding to dead gRNA and target) the adapter proteins bind and, the functional domain on the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective.
  • the functional domain is a transcription activator (e.g. VP64 or p65)
  • the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target.
  • a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g. Fokl) will be advantageously positioned to cleave or partially cleave the target.
  • the skilled person will understand that modifications to the dead gRNA which allow for binding of the adapter + functional domain but not proper positioning of the adapter + functional domain (e.g. due to steric hindrance within the three dimensional structure of the CRISPR complex) are modifications which are not intended.
  • the one or more modified dead gRNA may be modified at the tetra loop, the stem loop 1, stem loop 2, or stem loop 3, as described herein, preferably at either the tetra loop or stem loop 2, and most preferably at both the tetra loop and stem loop 2.
  • the functional domains may be, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible).
  • methylase activity demethylase activity
  • transcription activation activity e.g. transcription activation activity
  • transcription repression activity e.g. light inducible
  • transcription release factor activity e.g. light inducible
  • histone modification activity e.g. light inducible
  • RNA cleavage activity e.g. DNA cleavage activity
  • nucleic acid binding activity e.g. light inducible
  • molecular switches e.g. light inducible
  • the dead gRNA may be designed to include multiple binding recognition sites (e.g. aptamers) specific to the same or different adapter protein.
  • the dead gRNA may be designed to bind to the promoter region -1000 - +1 nucleic acids upstream of the transcription start site (i.e. TSS), preferably -200 nucleic acids. This positioning improves functional domains which affect gene activation (e.g. transcription activators) or gene inhibition (e.g. transcription repressors).
  • the modified dead gRNA may be one or more modified dead gRNAs targeted to one or more target loci (e.g. at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 gRNA, at least 50 gRNA) comprised in a composition.
  • the adaptor protein may be any number of proteins that binds to an aptamer or recognition site introduced into the modified dead gRNA and which allows proper positioning of one or more functional domains, once the dead gRNA has been incorporated into the CRISPR complex, to affect the target with the attributed function.
  • such may be coat proteins, preferably bacteriophage coat proteins.
  • the functional domains associated with such adaptor proteins e.g.
  • fusion protein in the form of fusion protein may include, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible).
  • Preferred domains are Fokl, VP64, P65, HSFl, MyoDl .
  • the functional domain is a transcription activator or transcription repressor it is advantageous that additionally at least an NLS is provided and preferably at the N terminus. When more than one functional domain is included, the functional domains may be the same or different.
  • the adaptor protein may utilize known linkers to attach such functional domains.
  • the modified dead gRNA, the (inactivated) Casl3 (with or without functional domains), and the binding protein with one or more functional domains may each individually be comprised in a composition and administered to a host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host. Administration to a host may be performed via viral vectors known to the skilled person or described herein for delivery to a host (e.g. lentiviral vector, adenoviral vector, AAV vector). As explained herein, use of different selection markers (e.g. for lentiviral gRNA selection) and concentration of gRNA (e.g. dependent on whether multiple gRNAs are used) may be advantageous for eliciting an improved effect.
  • compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g. gene activation of lincRNA and identification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes).
  • the current invention comprehends the use of the compositions of the current invention to establish and utilize conditional or inducible CRISPR transgenic cell /animals, which are not believed prior to the present invention or application.
  • the target cell comprises Casl 3 conditionally or inducibly (e.g. in the form of Cre dependent constructs) and/or the adapter protein conditionally or inducibly and, on expression of a vector introduced into the target cell, the vector expresses that which induces or gives rise to the condition of Casl3 expression and/or adaptor expression in the target cell.
  • CRISPR knock-in / conditional transgenic animal e.g. mouse comprising e.g. a Lox-Stop- polyA-Lox(LSL) cassette
  • one or more compositions providing one or more modified dead gRNA (e.g. -200 nucleotides to TSS of a target gene of interest for gene activation purposes) as described herein (e.g. modified dead gRNA with one or more aptamers recognized by coat proteins, e.g. MS2), one or more adapter proteins as described herein (MS2 binding protein linked to one or more VP64) and means for inducing the conditional animal (e.g.
  • modified dead gRNA e.g. -200 nucleotides to TSS of a target gene of interest for gene activation purposes
  • coat proteins e.g. MS2
  • adapter proteins as described herein (MS2 binding protein linked to one or more VP64)
  • means for inducing the conditional animal e.g.
  • the adaptor protein may be provided as a conditional or inducible element with a conditional or inducible Casl3 to provide an effective model for screening purposes, which advantageously only requires minimal design and administration of specific dead gRNAs for a broad number of applications.
  • a protected guide RNA comprises a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell and a protector strand, wherein the protector strand is optionally complementary to the guide sequence and wherein the guide sequence may in part be hybridizable to the protector strand.
  • the pgRNA optionally includes an extension sequence. The thermodynamics of the pgRNA-target DNA hybridization is determined by the number of bases complementary between the guide RNA and target DNA.
  • a protector sequence By employing 'thermodynamic protection', specificity of dead gRNA can be improved by adding a protector sequence. For example, one method adds a complementary protector strand of varying lengths to the 3 ' end of the guide sequence within the dead gRNA. As a result, the protector strand is bound to at least a portion of the dead gRNA and provides for a protected gRNA (pgRNA). In turn, the dead gRNA references herein may be easily protected using the described embodiments, resulting in pgRNA.
  • the protector strand can be either a separate RNA transcript or strand or a chimeric version joined to the 3' end of the dead gRNA guide sequence.
  • CRISPR enzymes as defined herein can employ more than one RNA guide without losing activity. This enables the use of the CRISPR enzymes, systems or complexes as defined herein for targeting multiple DNA targets, genes or gene loci, with a single enzyme, system or complex as defined herein.
  • the guide RNAs may be tandemly arranged, optionally separated by a nucleotide sequence such as a direct repeat as defined herein. The position of the different guide RNAs is the tandem does not influence the activity. It is noted that the terms "CRISPR-Cas system”, “CRISP-Cas complex” "CRISPR complex” and "CRISPR system” are used interchangeably.
  • CRISPR enzyme Cas enzyme
  • Cas enzyme CRISPR-Cas enzyme
  • said CRISPR enzyme, CRISP-Cas enzyme or Cas enzyme is Cas 13, or any one of the modified or mutated variants thereof described herein elsewhere.
  • the invention provides a non-naturally occurring or engineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferably a Type V or VI CRISPR enzyme as described herein, such as without limitation Cas 13 as described herein elsewhere, used for tandem or multiplex targeting.
  • a non-naturally occurring or engineered CRISPR enzyme preferably a class 2 CRISPR enzyme, preferably a Type V or VI CRISPR enzyme as described herein, such as without limitation Cas 13 as described herein elsewhere, used for tandem or multiplex targeting.
  • CRISPR or CRISPR-Cas or Cas
  • Any of the methods, products, compositions and uses as described herein elsewhere are equally applicable with the multiplex or tandem targeting approach further detailed below.
  • the invention provides for the use of a Casl3 enzyme, complex or system as defined herein for targeting multiple gene loci. In one embodiment, this can be established by using multiple (tandem or multiplex) guide RNA (gRNA) sequences.
  • gRNA guide RNA
  • the invention provides methods for using one or more elements of a Cas 13 enzyme, complex or system as defined herein for tandem or multiplex targeting, wherein said CRISP system comprises multiple guide RNA sequences.
  • said gRNA sequences are separated by a nucleotide sequence, such as a direct repeat as defined herein elsewhere.
  • the Cas 13 enzyme, system or complex as defined herein provides an effective means for modifying multiple target polynucleotides.
  • the Cas 13 enzyme, system or complex as defined herein has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) one or more target polynucleotides in a multiplicity of cell types.
  • the Cas 13 enzyme, system or complex as defined herein of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis, including targeting multiple gene loci within a single CRISPR system.
  • the invention provides a Casl3 enzyme, system or complex as defined herein, i.e. a Casl3 CRISPR-Cas complex having a Casl3 protein having at least one destabilization domain associated therewith, and multiple guide RNAs that target multiple nucleic acid molecules such as DNA molecules, whereby each of said multiple guide RNAs specifically targets its corresponding nucleic acid molecule, e.g., DNA molecule.
  • Each nucleic acid molecule target e.g., DNA molecule can encode a gene product or encompass a gene locus.
  • Using multiple guide RNAs hence enables the targeting of multiple gene loci or multiple genes.
  • the Casl3 enzyme may cleave the DNA molecule encoding the gene product.
  • expression of the gene product is altered.
  • the Casl3 protein and the guide RNAs do not naturally occur together.
  • the invention comprehends the guide RNAs comprising tandemly arranged guide sequences.
  • the invention further comprehends coding sequences for the Cas 13 protein being codon optimized for expression in a eukaryotic cell .
  • the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell and in a more preferred embodiment the mammalian cell is a human cell. Expression of the gene product may be decreased.
  • the Cas 13 enzyme may form part of a CRISPR system or complex, which further comprises tandemly arranged guide RNAs (gRNAs) comprising a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 25, 30, or more than 30 guide sequences, each capable of specifically hybridizing to a target sequence in a genomic locus of interest in a cell.
  • gRNAs tandemly arranged guide RNAs
  • the functional Cas 13 CRISPR system or complex binds to the multiple target sequences.
  • the functional CRISPR system or complex may edit the multiple target sequences, e.g., the target sequences may comprise a genomic locus, and in some embodiments there may be an alteration of gene expression.
  • the functional CRISPR system or complex may comprise further functional domains.
  • the invention provides a method for altering or modifying expression of multiple gene products.
  • the method may comprise introducing into a cell containing said target nucleic acids, e.g., DNA molecules, or containing and expressing target nucleic acid, e.g., DNA molecules; for instance, the target nucleic acids may encode gene products or provide for expression of gene products (e.g., regulatory sequences).
  • the Cas 13 enzyme used for multiplex targeting is associated with one or more functional domains.
  • the CRISPR enzyme used for multiplex targeting is a deadCasl3 as defined herein elsewhere.
  • the present invention provides a means for delivering the Cas 13 enzyme, system or complex for use in multiple targeting as defined herein or the polynucleotides defined herein.
  • delivery means are e.g. particle(s) delivering component(s) of the complex, vector(s) comprising the polynucleotide(s) discussed herein (e.g., encoding the CRISPR enzyme, providing the nucleotides encoding the CRISPR complex).
  • the vector may be a plasmid or a viral vector such as AAV, or lentivirus. Transient transfection with plasmids, e.g., into HEK cells may be advantageous, especially given the size limitations of AAV and that while Casl3 fits into AAV, one may reach an upper limit with additional guide RNAs.
  • a model that constitutively expresses the Casl3 enzyme, complex or system as used herein for use in multiplex targeting.
  • the organism may be transgenic and may have been transfected with the present vectors or may be the offspring of an organism so transfected.
  • the present invention provides compositions comprising the CRISPR enzyme, system and complex as defined herein or the polynucleotides or vectors described herein.
  • Casl3 CRISPR systems or complexes comprising multiple guide RNAs, preferably in a tandemly arranged format. Said different guide RNAs may be separated by nucleotide sequences such as direct repeats.
  • a method of treating a subject comprising inducing gene editing by transforming the subject with the polynucleotide encoding the Casl3 CRISPR system or complex or any of polynucleotides or vectors described herein and administering them to the subject.
  • a suitable repair template may also be provided, for example delivered by a vector comprising said repair template.
  • a method of treating a subject comprising inducing transcriptional activation or repression of multiple target gene loci by transforming the subject with the polynucleotides or vectors described herein, wherein said polynucleotide or vector encodes or comprises the Casl3 enzyme, complex or system comprising multiple guide RNAs, preferably tandemly arranged.
  • a subject e.g., a subject in need thereof
  • said polynucleotide or vector encodes or comprises the Casl3 enzyme, complex or system comprising multiple guide RNAs, preferably tandemly arranged.
  • compositions comprising Casl3 enzyme, complex or system comprising multiple guide RNAs, preferably tandemly arranged, or the polynucleotide or vector encoding or comprising said Casl3 enzyme, complex or system comprising multiple guide RNAs, preferably tandemly arranged, for use in the methods of treatment as defined herein elsewhere are also provided.
  • a kit of parts may be provided including such compositions.
  • Use of said composition in the manufacture of a medicament for such methods of treatment are also provided.
  • Use of a Casl3 CRISPR system in screening is also provided by the present invention, e.g., gain of function screens.
  • Cells which are artificially forced to overexpress a gene are be able to down regulate the gene over time (re-establishing equilibrium) e.g. by negative feedback loops. By the time the screen starts the unregulated gene might be reduced again.
  • Using an inducible Casl3 activator allows one to induce transcription right before the screen and therefore minimizes the chance of false negative hits. Accordingly, by use of the instant invention in screening, e.g., gain of function screens, the chance of false negative results may be minimized.
  • the invention provides an engineered, non-naturally occurring CRISPR system comprising a Casl3 protein and multiple guide R As that each specifically target a DNA molecule encoding a gene product in a cell, whereby the multiple guide RNAs each target their specific DNA molecule encoding the gene product and the Casl3 protein cleaves the target DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the CRISPR protein and the guide RNAs do not naturally occur together.
  • the invention comprehends the multiple guide RNAs comprising multiple guide sequences, preferably separated by a nucleotide sequence such as a direct repeat and optionally fused to a tracr sequence.
  • the CRISPR protein is a type V or VI CRISPR-Cas protein and in a more preferred embodiment the CRISPR protein is a Casl3 protein.
  • the invention further comprehends a Casl3 protein being codon optimized for expression in a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell.
  • the expression of the gene product is decreased.
  • the invention provides an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to the multiple Casl3 CRISPR system guide RNAs that each specifically target a DNA molecule encoding a gene product and a second regulatory element operably linked coding for a CRISPR protein. Both regulatory elements may be located on the same vector or on different vectors of the system.
  • the multiple guide RNAs target the multiple DNA molecules encoding the multiple gene products in a cell and the CRISPR protein may cleave the multiple DNA molecules encoding the gene products (it may cleave one or both strands or have substantially no nuclease activity), whereby expression of the multiple gene products is altered; and, wherein the CRISPR protein and the multiple guide RNAs do not naturally occur together.
  • the CRISPR protein is Casl3 protein, optionally codon optimized for expression in a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell and in a more preferred embodiment the mammalian cell is a human cell.
  • the expression of each of the multiple gene products is altered, preferably decreased.
  • the invention provides a vector system comprising one or more vectors.
  • the system comprises: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the one or more guide sequence(s) direct(s) sequence-specific binding of the CRISPR complex to the one or more target sequence(s) in a eukaryotic cell, wherein the CRISPR complex comprises a Casl3 enzyme complexed with the one or more guide sequence(s) that is hybridized to the one or more target sequence(s); and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Casl3 enzyme, preferably comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on the same or different vectors of the system.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Casl3 CRISPR complex to a different target sequence in a eukaryotic cell.
  • the CRISPR complex comprises one or more nuclear localization sequences and/or one or more NES of sufficient strength to drive accumulation of said Casl3 CRISPR complex in a detectable amount in or out of the nucleus of a eukaryotic cell.
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length.
  • Recombinant expression vectors can comprise the polynucleotides encoding the Casl3 enzyme, system or complex for use in multiple targeting as defined herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • a host cell is transiently or non-transiently transfected with one or more vectors comprising the polynucleotides encoding the Casl3 enzyme, system or complex for use in multiple targeting as defined herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art and exemplified herein elsewhere.
  • a cell transfected with one or more vectors comprising the polynucleotides encoding the Casl3 enzyme, system or complex for use in multiple targeting as defined herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the components of a Casl3 CRISPR system or complex for use in multiple targeting as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a Cas 13 CRISPR system or complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • cells transiently or non-transiently transfected with one or more vectors comprising the polynucleotides encoding the Casl3 enzyme, system or complex for use in multiple targeting as defined herein, or cell lines derived from such cells are used in assessing one or more test compounds.
  • regulatory element is as defined herein elsewhere.
  • Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.
  • the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence(s) direct(s) sequence-specific binding of the Casl3 CRISPR complex to the respective target sequence(s) in a eukaryotic cell, wherein the Casl3 CRISPR complex comprises a Casl3 enzyme complexed with the one or more guide sequence(s) that is hybridized to the respective target sequence(s); and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Casl3 enzyme comprising preferably at least one nuclear localization sequence and/or NES.
  • the host cell comprises components (a) and (b). Where applicable, a tracr sequence may also be provided.
  • component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, and optionally separated by a direct repeat, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Casl3 CRISPR complex to a different target sequence in a eukaryotic cell.
  • the Casl3 enzyme comprises one or more nuclear localization sequences and/or nuclear export sequences or NES of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in and/or out of the nucleus of a eukaryotic cell.
  • the Casl3 enzyme is a type V or VI CRISPR system enzyme. In some embodiments, the Casl3 enzyme is a Casl3 enzyme. In some embodiments, the Casl3 enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 1_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.
  • the Cas 13 enzyme is codon-optimized for expression in a eukaryotic cell.
  • the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the one or more guide sequence(s) is (are each) at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16- 20 nucleotides in length. When multiple guide R As are used, they are preferably separated by a direct repeat sequence.
  • the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments.
  • the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments.
  • the organism in some embodiments of these aspects may be an animal; for example a mammal. Also, the organism may be an arthropod such as an insect. The organism also may be a plant. Further, the organism may be a fungus.
  • the invention provides a kit comprising one or more of the components described herein.
  • the kit comprises a vector system and instructions for using the kit.
  • the vector system comprises (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Casl3 CRISPR complex to a target sequence in a eukaryotic cell, wherein the Casl3 CRISPR complex comprises a Casl3 enzyme complexed with the guide sequence that is hybridized to the target sequence; and/or (b) a second regulatory element operably linked to an enzyme- coding sequence encoding said Casl3 enzyme comprising a nuclear localization sequence.
  • a tracr sequence may also be provided.
  • the kit comprises components (a) and (b) located on the same or different vectors of the system.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell.
  • the Casl3 enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.
  • the CRISPR enzyme is a type V or VI CRISPR system enzyme.
  • the CRISPR enzyme is a Casl3 enzyme.
  • the Casl3 enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.
  • the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.
  • the DD-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.
  • the DD-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity).
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length.
  • the invention provides a method of modifying multiple target polynucleotides in a host cell such as a eukaryotic cell.
  • the method comprises allowing a Cas 13CRISPR complex to bind to multiple target polynucleotides, e.g., to effect cleavage of said multiple target polynucleotides, thereby modifying multiple target polynucleotides, wherein the Casl3CRISPR complex comprises a Cas 13 enzyme complexed with multiple guide sequences each of the being hybridized to a specific target sequence within said target polynucleotide, wherein said multiple guide sequences are linked to a direct repeat sequence.
  • a tracr sequence may also be provided (e.g. to provide a single guide RNA, sgRNA).
  • said cleavage comprises cleaving one or two strands at the location of each of the target sequence by said Cas 13 enzyme.
  • said cleavage results in decreased transcription of the multiple target genes.
  • the method further comprises repairing one or more of said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of one or more of said target polynucleotides.
  • said mutation results in one or more amino acid changes in a protein expressed from a gene comprising one or more of the target sequence(s).
  • the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the Cas 13 enzyme and the multiple guide RNA sequence linked to a direct repeat sequence. Where applicable, a tracr sequence may also be provided.
  • said vectors are delivered to the eukaryotic cell in a subject.
  • said modifying takes place in said eukaryotic cell in a cell culture.
  • the method further comprises isolating said eukaryotic cell from a subject prior to said modifying.
  • the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.
  • the invention provides a method of modifying expression of multiple polynucleotides in a eukaryotic cell.
  • the method comprises allowing a Cas 13 CRISPR complex to bind to multiple polynucleotides such that said binding results in increased or decreased expression of said polynucleotides; wherein the Casl3 CRISPR complex comprises a Casl3 enzyme complexed with multiple guide sequences each specifically hybridized to its own target sequence within said polynucleotide, wherein said guide sequences are linked to a direct repeat sequence.
  • a tracr sequence may also be provided.
  • the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the Cas 13 enzyme and the multiple guide sequences linked to the direct repeat sequences .
  • a tracr sequence may also be provided.
  • the invention provides a recombinant polynucleotide comprising multiple guide RNA sequences up- or downstream (whichever applicable) of a direct repeat sequence, wherein each of the guide sequences when expressed directs sequence-specific binding of a Cas 13 CRISPR complex to its corresponding target sequence present in a eukaryotic cell.
  • the target sequence is a viral sequence present in a eukaryotic cell. Where applicable, a tracr sequence may also be provided.
  • the target sequence is a proto-oncogene or an oncogene.
  • aspects of the invention encompass a non-naturally occurring or engineered composition that may comprise a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell and a Cas 13 enzyme as defined herein that may comprise at least one or more nuclear localization sequences.
  • gRNA guide RNA
  • Cas 13 enzyme as defined herein that may comprise at least one or more nuclear localization sequences.
  • An aspect of the invention encompasses methods of modifying a genomic locus of interest to change gene expression in a cell by introducing into the cell any of the compositions described herein.
  • An aspect of the invention is that the above elements are comprised in a single composition or comprised in individual compositions. These compositions may advantageously be applied to a host to elicit a functional effect on the genomic level.
  • the term "guide RNA” or "gRNA” has the leaning as used herein elsewhere and comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • Each gRNA may be designed to include multiple binding recognition sites (e.g., aptamers) specific to the same or different adapter protein.
  • Each gRNA may be designed to bind to the promoter region - 1000 - + 1 nucleic acids upstream of the transcription start site (i.e. TSS), preferably -200 nucleic acids.
  • the modified gRNA may be one or more modified gRNAs targeted to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a composition.
  • target loci e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA
  • Said multiple gRNA sequences can be tandemly arranged and are preferably separated by a direct repeat.
  • the CRISPR enzyme as defined herein may each individually be comprised in a composition and administered to a host individually or collectively.
  • these components may be provided in a single composition for administration to a host.
  • Administration to a host may be performed via viral vectors known to the skilled person or described herein for delivery to a host (e.g., lentiviral vector, adenoviral vector, AAV vector).
  • viral vectors known to the skilled person or described herein for delivery to a host (e.g., lentiviral vector, adenoviral vector, AAV vector).
  • viral vectors e.g., lentiviral vector, adenoviral vector, AAV vector.
  • selection markers e.g., for lentiviral sgRNA selection
  • concentration of gRNA e.g., dependent on whether multiple gRNAs are used
  • compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g., gene activation of lincRNA and identification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes).
  • the current invention comprehends the use of the compositions of the current invention to establish and utilize conditional or inducible CRISPR transgenic cell /animals; see, e.g., Piatt et al., Cell (2014), 159(2): 440-455, or PCT patent publications cited herein, such as WO 2014/093622 (PCT/US2013/074667).
  • cells or animals such as non-human animals, e.g., vertebrates or mammals, such as rodents, e.g., mice, rats, or other laboratory or field animals, e.g., cats, dogs, sheep, etc., may be 'knock-in' whereby the animal conditionally or inducibly expresses Casl3 akin to Piatt et al.
  • rodents e.g., mice, rats, or other laboratory or field animals, e.g., cats, dogs, sheep, etc.
  • the target cell or animal thus comprises the CRISPR enzyme (e.g., Casl3) conditionally or inducibly (e.g., in the form of Cre dependent constructs), on expression of a vector introduced into the target cell, the vector expresses that which induces or gives rise to the condition of the CRISPR enzyme (e.g., Casl3) expression in the target cell.
  • CRISPR enzyme e.g., Casl3
  • inducible genomic events are also an aspect of the current invention. Examples of such inducible events have been described herein elsewhere.
  • phenotypic alteration is preferably the result of genome modification when a genetic disease is targeted, especially in methods of therapy and preferably where a repair template is provided to correct or alter the phenotype.
  • diseases that may be targeted include those concerned with disease- causing splice defects.
  • cellular targets include Hemopoietic Stem/Progenitor Cells (CD34+); Human T cells; and Eye (retinal cells) - for example photoreceptor precursor cells.
  • CD34+ Hemopoietic Stem/Progenitor Cells
  • Human T cells Human T cells
  • Eye (retinal cells) for example photoreceptor precursor cells.
  • Gene targets include: Human Beta Globin - HBB (for treating Sickle Cell Anemia, including by stimulating gene-conversion (using closely related HBD gene as an endogenous template)); CD3 (T-Cells); and CEP920 - retina (eye).
  • disease targets also include: cancer; Sickle Cell Anemia (based on a point mutation); HBV, HIV; Beta-Thalassemia; and ophthalmic or ocular disease - for example Leber Congenital Amaurosis (LCA)-causing Splice Defect.
  • cancer Sickle Cell Anemia (based on a point mutation); HBV, HIV; Beta-Thalassemia; and ophthalmic or ocular disease - for example Leber Congenital Amaurosis (LCA)-causing Splice Defect.
  • CUA Leber Congenital Amaurosis
  • delivery methods include: Cationic Lipid Mediated “direct” delivery of Enzyme- Guide complex (RiboNucleoProtein) and electroporation of plasmid DNA.
  • any of the methods described herein may be applied in vitro and ex vivo.
  • non-naturally occurring or engineered composition comprising:
  • the first and the second guide sequences direct sequence- specific binding of a first and a second Casl3 CRISPR complex to the first and second target sequences respectively,
  • first CRISPR complex comprises the Casl3 enzyme complexed with the first guide sequence that is hybridizable to the first target sequence
  • second CRISPR complex comprises the Casl3 enzyme complexed with the second guide sequence that is hybridizable to the second target sequence
  • compositions comprising more than two guide RNAs can be envisaged e.g. each specific for one target, and arranged tandemly in the composition or CRISPR system or complex as described herein.
  • the Casl3 is delivered into the cell as a protein.
  • the Casl3 is delivered into the cell as a protein or as a nucleotide sequence encoding it. Delivery to the cell as a protein may include delivery of a Ribonucleoprotein (RNP) complex, where the protein is complexed with the multiple guides.
  • RNP Ribonucleoprotein
  • host cells and cell lines modified by or comprising the compositions, systems or modified enzymes of present invention are provided, including stem cells, and progeny thereof.
  • methods of cellular therapy are provided, where, for example, a single cell or a population of cells is sampled or cultured, wherein that cell or cells is or has been modified ex vivo as described herein, and is then re-introduced (sampled cells) or introduced (cultured cells) into the organism.
  • Stem cells whether embryonic or induce pluripotent or totipotent stem cells, are also particularly preferred in this regard. But, of course, in vivo embodiments are also envisaged.
  • Inventive methods can further comprise delivery of templates, such as repair templates, which may be dsODN or ssODN, see below.
  • Delivery of templates may be via the cotemporaneous or separate from delivery of any or all the CRISPR enzyme or guide RNAs and via the same delivery mechanism or different.
  • it is preferred that the template is delivered together with the guide RNAs and, preferably, also the CRISPR enzyme.
  • An example may be an AAV vector where the CRISPR enzyme is AsCasl3 or LbCasl3.
  • Inventive methods can further comprise: (a) delivering to the cell a double-stranded oligodeoxynucleotide (dsODN) comprising overhangs complimentary to the overhangs created by said double strand break, wherein said dsODN is integrated into the locus of interest; or -(b) delivering to the cell a single-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template for homology directed repair of said double strand break.
  • Inventive methods can be for the prevention or treatment of disease in an individual, optionally wherein said disease is caused by a defect in said locus of interest.
  • Inventive methods can be conducted in vivo in the individual or ex vivo on a cell taken from the individual, optionally wherein said cell is returned to the individual.
  • the invention also comprehends products obtained from using CRISPR enzyme or Cas enzyme or Casl3 enzyme or CRISPR-CRISPR enzyme or CRISPR-Cas system or CRISPR-Casl3 system for use in tandem or multiple targeting as defined herein.
  • the invention provides escorted Cas 13 CRISPR-Cas systems or complexes, especially such a system involving an escorted Cas 13 CRISPR-Cas system guide.
  • escorted is meant that the Cas 13 CRISPR-Cas system or complex or guide is delivered to a selected time or place within a cell, so that activity of the Cas 13 CRISPR-Cas system or complex or guide is spatially or temporally controlled.
  • the activity and destination of the Cas 13 CRISPR-Cas system or complex or guide may be controlled by an escort RNA aptamer sequence that has binding affinity for an aptamer ligand, such as a cell surface protein or other localized cellular component.
  • the escort aptamer may for example be responsive to an aptamer effector on or in the cell, such as a transient effector, such as an external energy source that is applied to the cell at a particular time.
  • the escorted Cas 13 CRISPR-Cas systems or complexes have a gRNA with a functional structure designed to improve gRNA structure, architecture, stability, genetic expression, or any combination thereof.
  • a structure can include an aptamer.
  • Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990, 249:505-510).
  • Nucleic acid aptamers can for example be selected from pools of random-sequence oligonucleotides, with high binding affinities and specificities for a wide range of biomedically relevant targets, suggesting a wide range of therapeutic utilities for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington.
  • aptamers as therapeutics. Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a wide range of uses for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. "Nanotechnology and aptamers: applications in drug delivery.” Trends in biotechnology 26.8 (2008): 442-449; and, Hicke BJ, Stephens AW. "Escort aptamers: a delivery service for diagnosis and therapy.” J Clin Invest 2000, 106:923-928.).
  • RNA aptamers may also be constructed that function as molecular switches, responding to a que by changing properties, such as RNA aptamers that bind fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Sarnie R. Jaffrey. "RNA mimics of green fluorescent protein.” Science 333.6042 (2011): 642-646). It has also been suggested that aptamers may be used as components of targeted siRNA therapeutic delivery systems, for example targeting cell surface proteins (Zhou, Jiehua, and John J. Rossi. "Aptamer-targeted cell-specific RNA interference.” Silence 1.1 (2010): 4).
  • a gR A modified, e.g., by one or more aptamer(s) designed to improve gRNA delivery, including delivery across the cellular membrane, to intracellular compartments, or into the nucleus.
  • a structure can include, either in addition to the one or more aptamer(s) or without such one or more aptamer(s), moiety(ies) so as to render the guide deliverable, inducible or responsive to a selected effector.
  • the invention accordingly comprehends an gRNA that responds to normal or pathological physiological conditions, including without limitation pH, hypoxia, O2 concentration, temperature, protein concentration, enzymatic concentration, lipid structure, light exposure, mechanical disruption (e.g. ultrasound waves), magnetic fields, electric fields, or electromagnetic radiation.
  • An aspect of the invention provides non-naturally occurring or engineered composition
  • egRNA escorted guide RNA
  • RNA guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell
  • an escort RNA aptamer sequence wherein the escort aptamer has binding affinity for an aptamer ligand on or in the cell, or the escort aptamer is responsive to a localized aptamer effector on or in the cell, wherein the presence of the aptamer ligand or effector on or in the cell is spatially or temporally restricted.
  • the escort aptamer may for example change conformation in response to an interaction with the aptamer ligand or effector in the cell.
  • the escort aptamer may have specific binding affinity for the aptamer ligand.
  • the aptamer ligand may be localized in a location or compartment of the cell, for example on or in a membrane of the cell. Binding of the escort aptamer to the aptamer ligand may accordingly direct the egRNA to a location of interest in the cell, such as the interior of the cell by way of binding to an aptamer ligand that is a cell surface ligand. In this way, a variety of spatially restricted locations within the cell may be targeted, such as the cell nucleus or mitochondria.
  • the self inactivating Casl3 CRISPR-Cas system includes additional RNA (i.e., guide RNA) that targets the coding sequence for the CRISPR enzyme itself or that targets one or more non- coding guide target sequences complementary to unique sequences present in one or more of the following: (a) within the promoter driving expression of the non-coding RNA elements, (b) within the promoter driving expression of the Cas 13 gene, (c) within lOObp of the ATG translational start codon in the Casl3 coding sequence, (d) within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in an AAV genome.
  • guide RNA RNA that targets the coding sequence for the CRISPR enzyme itself or that targets one or more non- coding guide target sequences complementary to unique sequences present in one or more of the following: (a) within the promoter driving expression of the non-coding RNA elements, (b) within the promoter driving expression of the Cas 13 gene, (c) within l
  • the egRNA may include an RNA aptamer linking sequence, operably linking the escort RNA sequence to the RNA guide sequence.
  • the egRNA may include one or more photolabile bonds or non-naturally occurring residues.
  • the escort RNA aptamer sequence may be complementary to a target miRNA, which may or may not be present within a cell, so that only when the target miRNA is present is there binding of the escort RNA aptamer sequence to the target miRNA which results in cleavage of the egRNA by an RNA-induced silencing complex (RISC) within the cell.
  • RISC RNA-induced silencing complex
  • the escort RNA aptamer sequence may for example be from 10 to 200 nucleotides in length, and the egRNA may include more than one escort RNA aptamer sequence.
  • 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 certain embodiments the guide RNA or mature crRNA comprises 19 nts of partial direct repeat followed by 23-25 nt of guide sequence or spacer sequence.
  • the effector protein is a FnCasl3 effector protein and requires at least 16 nt of guide sequence to achieve detectable DNA cleavage and a minimum of 17 nt of guide sequence to achieve efficient DNA cleavage in vitro.
  • the direct repeat sequence is located upstream (i.e., 5') from the guide sequence or spacer sequence.
  • the seed sequence (i.e. the sequence essential critical for recognition and/or hybridization to the sequence at the target locus) of theCasl3Casl3 guide RNA is approximately within the first 5 nt on the 5' end of the guide sequence or spacer sequence.
  • the egRNA may be included in a non-naturally occurring or engineered Cas 13 Cas 13 CRISPR- Cas complex composition, together with a Cas 13 which may include at least one mutation, for example a mutation so that the Cas 13 has no more than 5% of the nuclease activity of a Cas 13 not having the at least one mutation, for example having a diminished nuclease activity of at least 97%, or 100% as compared with the Cas 13 not having the at least one mutation.
  • the Cas 13 may also include one or more nuclear localization sequences. Mutated Casl3 enzymes having modulated activity such as diminished nuclease activity are described herein elsewhere.
  • the engineered Casl3 CRISPR-Cas composition may be provided in a cell, such as a eukaryotic cell, a mammalian cell, or a human cell.
  • compositions described herein comprise a Casl3 CRISPR-Cas complex having at least three functional domains, at least one of which is associated with Casl3 and at least two of which are associated with egRNA.
  • compositions described herein may be used to introduce a genomic locus event in a host cell, such as an eukaryotic cell, in particular a mammalian cell, or a non-human eukaryote, in particular a non-human mammal such as a mouse, in vivo.
  • the genomic locus event may comprise affecting gene activation, gene inhibition, or cleavage in a locus.
  • the compositions described herein may also be used to modify a genomic locus of interest to change gene expression in a cell. Methods of introducing a genomic locus event in a host cell using the Casl3 enzyme provided herein are described herein in detail elsewhere.
  • Delivery of the composition may for example be by way of delivery of a nucleic acid molecule(s) coding for the composition, which nucleic acid molecule(s) is operatively linked to regulatory sequence(s), and expression of the nucleic acid molecule(s) in vivo, for example by way of a lentivirus, an adenovirus, or an AAV.
  • the present invention provides compositions and methods by which gRNA-mediated gene editing activity can be adapted.
  • the invention provides gRNA secondary structures that improve cutting efficiency by increasing gRNA and/or increasing the amount of RNA delivered into the cell.
  • the gRNA may include light labile or inducible nucleotides.
  • gRNA for example gRNA delivered with viral or non-viral technologies
  • Applicants added secondary structures into the gRNA that enhance its stability and improve gene editing.
  • Applicants modified gRNAs with cell penetrating RNA aptamers; the aptamers bind to cell surface receptors and promote the entry of gRNAs into cells.
  • the cell-penetrating aptamers can be designed to target specific cell receptors, in order to mediate cell-specific delivery.
  • Applicants also have created guides that are inducible.
  • Light responsiveness of an inducible system may be achieved via the activation and binding of cryptochrome-2 and CIB 1.
  • Blue light stimulation induces an activating conformational change in cryptochrome-2, resulting in recruitment of its binding partner CIB 1.
  • This binding is fast and reversible, achieving saturation in ⁇ 15 sec following pulsed stimulation and returning to baseline ⁇ 15 min after the end of stimulation.
  • Crytochrome-2 activation is also highly sensitive, allowing for the use of low light intensity stimulation and mitigating the risks of phototoxicity. Further, in a context such as the intact mammalian brain, variable light intensity may be used to control the size of a stimulated region, allowing for greater precision than vector delivery alone may offer.
  • the invention contemplates energy sources such as electromagnetic radiation, sound energy or thermal energy to induce the guide.
  • the electromagnetic radiation is a component of visible light.
  • the light is a blue light with a wavelength of about 450 to about 495 nm.
  • the wavelength is about 488 nm.
  • the light stimulation is via pulses.
  • the light power may range from about 0-9 mW/cm 2 .
  • a stimulation paradigm of as low as 0.25 sec every 15 sec should result in maximal activation.
  • Cells involved in the practice of the present invention may be a prokaryotic cell or a eukaryotic cell, advantageously an animal cell a plant cell or a yeast cell, more advantageously a mammalian cell.
  • the chemical or energy sensitive guide may undergo a conformational change upon induction by the binding of a chemical source or by the energy allowing it act as a guide and have the Cas 13 CRISPR- Cas system or complex function.
  • the invention can involve applying the chemical source or energy so as to have the guide function and the Cas 13 CRISPR-Cas system or complex function; and optionally further determining that the expression of the genomic locus is altered.
  • ABI-PYL based system inducible by Abscisic Acid (ABA) see, e.g., http://stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2
  • FKBP-FRB based system inducible by rapamycin or related chemicals based on rapamycin
  • GID l-GAI based system inducible by Gibberellin GA
  • Another system contemplated by the present invention is a chemical inducible system based on change in sub-cellular localization.
  • the polypeptide include a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest linked to at least one or more effector domains are further linker to a chemical or energy sensitive protein.
  • TALE Transcription activator-like effector
  • This type of system could also be used to induce the cleavage of a genomic locus of interest in a cell when the effector domain is a nuclease.
  • a chemical inducible system can be an estrogen receptor (ER) based system inducible by 4- hydroxytamoxifen (40HT) (see, e.g., http://www.pnas.Org/content/104/3/1027.abstract).
  • ER estrogen receptor
  • 40HT 4- hydroxytamoxifen
  • a mutated ligand- binding domain of the estrogen receptor called ERT2 translocates into the nucleus of cells upon binding of 4-hydroxytamoxifen.
  • any naturally occurring or engineered derivative of any nuclear receptor, thyroid hormone receptor, retinoic acid receptor, estrogen receptor, estrogen-related receptor, glucocorticoid receptor, progesterone receptor, androgen receptor may be used in inducible systems analogous to the ER based inducible system.
  • TRP Transient receptor potential
  • This influx of ions will bind to intracellular ion interacting partners linked to a polypeptide including the guide and the other components of the Cas l3 CRISPR-Cas complex or system, and the binding will induce the change of sub-cellular localization of the polypeptide, leading to the entire polypeptide entering the nucleus of cells.
  • the guide protein and the other components of the Cas l3 CRISPR-Cas complex will be active and modulating target gene expression in cells.
  • This type of system could also be used to induce the cleavage of a genomic locus of interest in a cell; and, in this regard, it is noted that the Cas l3 enzyme is a nuclease.
  • the light could be generated with a laser or other forms of energy sources.
  • the heat could be generated by raise of temperature results from an energy source, or from nano-particles that release heat after absorbing energy from an energy source delivered in the form of radio-wave.
  • light activation may be an advantageous embodiment, sometimes it may be disadvantageous especially for in vivo applications in which the light may not penetrate the skin or other organs.
  • other methods of energy activation are contemplated, in particular, electric field energy and/or ultrasound which have a similar effect.
  • Electric field energy is preferably administered substantially as described in the art, using one or more electric pulses of from about 1 Volt/cm to about 10 kVolts/cm under in vivo conditions.
  • the electric field may be delivered in a continuous manner.
  • the electric pulse may be applied for between 1 and 500 milliseconds, preferably between 1 and 100 milliseconds.
  • the electric field may be applied continuously or in a pulsed manner for 5 about minutes.
  • 'electric field energy' is the electrical energy to which a cell is exposed.
  • the electric field has a strength of from about 1 Volt/cm to about 10 kVolts/cm or more under in vivo conditions (see WO97/49450).
  • the term "electric field” includes one or more pulses at variable capacitance and voltage and including exponential and/or square wave and/or modulated wave and/or modulated square wave forms. References to electric fields and electricity should be taken to include reference the presence of an electric potential difference in the environment of a cell. Such an environment may be set up by way of static electricity, alternating current (AC), direct current (DC), etc, as known in the art.
  • the electric field may be uniform, non-uniform or otherwise, and may vary in strength and/or direction in a time dependent manner.
  • Single or multiple applications of electric field, as well as single or multiple applications of ultrasound are also possible, in any order and in any combination.
  • the ultrasound and/or the electric field may be delivered as single or multiple continuous applications, or as pulses (pulsatile delivery).
  • Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells.
  • a sample of live cells is first mixed with the agent of interest and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture.
  • Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat. No 5,869,326).
  • the known electroporation techniques function by applying a brief high voltage pulse to electrodes positioned around the treatment region.
  • the electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the agent of interest enter the cells.
  • this electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 .mu.s duration.
  • Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820.
  • the electric field has a strength of from about 1 V/cm to about 10 kV/cm under in vitro conditions.
  • the electric field may have a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7 V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300 V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1 kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more.
  • the electric field has a strength of from about 1 V/cm to about 10 kV/cm under in vivo conditions.
  • the electric field strengths may be lowered where the number of pulses delivered to the target site are increased.
  • pulsatile delivery of electric fields at lower field strengths is envisaged.
  • the application of the electric field is in the form of multiple pulses such as double pulses of the same strength and capacitance or sequential pulses of varying strength and/or capacitance.
  • pulse includes one or more electric pulses at variable capacitance and voltage and including exponential and/or square wave and/or modulated wave/square wave forms.
  • the electric pulse is delivered as a waveform selected from an exponential wave form, a square wave form, a modulated wave form and a modulated square wave form.
  • a preferred embodiment employs direct current at low voltage.
  • Applicants disclose the use of an electric field which is applied to the cell, tissue or tissue mass at a field strength of between 1 V/cm and 20V/cm, for a period of 100 milliseconds or more, preferably 15 minutes or more.
  • Ultrasound is advantageously administered at a power level of from about 0.05 W/cm 2 to about
  • Diagnostic or therapeutic ultrasound may be used, or combinations thereof.
  • ultrasonic refers to a form of energy which consists of mechanical vibrations the frequencies of which are so high they are above the range of human hearing. Lower frequency limit of the ultrasonic spectrum may generally be taken as about 20 kHz. Most diagnostic applications of ultrasound employ frequencies in the range 1 and 15 MHz' (From Ultrasonics in Clinical Diagnosis, P. N.
  • Ultrasound has been used in both diagnostic and therapeutic applications.
  • diagnostic ultrasound ultrasound is typically used in an energy density range of up to about 100 mW/cm 2 (FDA recommendation), although energy densities of up to 750 mW/cm 2 have been used.
  • physiotherapy ultrasound is typically used as an energy source in a range up to about 3 to 4 W/cm 2
  • ultrasound In other therapeutic applications, higher intensities of ultrasound may be employed, for example, HIFU at 100 W/cm up to 1 kW/cm 2 (or even higher) for short periods of time.
  • HIFU at 100 W/cm up to 1 kW/cm 2 (or even higher) for short periods of time.
  • ultrasound as used in this specification is intended to encompass diagnostic, therapeutic and focused ultrasound.
  • Focused ultrasound allows thermal energy to be delivered without an invasive probe (see Morocz et al 1998 Journal of Magnetic Resonance Imaging Vol.8, No. 1, pp.136-142.
  • Another form of focused ultrasound is high intensity focused ultrasound (HIFU) which is reviewed by Moussatov et al in Ultrasonics (1998) Vol.36, No.8, pp.893-900 and TranHuuHue et al in Acustica (1997) Vol.83, No.6, pp.1103-1106.
  • a combination of diagnostic ultrasound and a therapeutic ultrasound is employed.
  • This combination is not intended to be limiting, however, and the skilled reader will appreciate that any variety of combinations of ultrasound may be used. Additionally, the energy density, frequency of ultrasound, and period of exposure may be varied.
  • the exposure to an ultrasound energy source is at a power density of from about 0.05 to about 100 Wcm "2 . Even more preferably, the exposure to an ultrasound energy source is at a power density of from about 1 to about 15 Wcm "2 .
  • the exposure to an ultrasound energy source is at a frequency of from about 0.015 to about 10.0 MHz. More preferably the exposure to an ultrasound energy source is at a frequency of from about 0.02 to about 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound is applied at a frequency of 3 MHz.
  • the exposure is for periods of from about 10 milliseconds to about 60 minutes. Preferably the exposure is for periods of from about 1 second to about 5 minutes. More preferably, the ultrasound is applied for about 2 minutes. Depending on the particular target cell to be disrupted, however, the exposure may be for a longer duration, for example, for 15 minutes.
  • the target tissue is exposed to an ultrasound energy source at an acoustic power density of from about 0.05 Wcm “2 to about 10 Wcm “2 with a frequency ranging from about 0.015 to about 10 MHz (see WO 98/52609).
  • an ultrasound energy source at an acoustic power density of above 100 Wcm "2 , but for reduced periods of time, for example, 1000 Wcm "2 for periods in the millisecond range or less.
  • the application of the ultrasound is in the form of multiple pulses; thus, both continuous wave and pulsed wave (pulsatile delivery of ultrasound) may be employed in any combination.
  • continuous wave ultrasound may be applied, followed by pulsed wave ultrasound, or vice versa. This may be repeated any number of times, in any order and combination.
  • the pulsed wave ultrasound may be applied against a background of continuous wave ultrasound, and any number of pulses may be used in any number of groups.
  • the ultrasound may comprise pulsed wave ultrasound.
  • the ultrasound is applied at a power density of 0.7 Wcm “2 or 1.25 Wcm "2 as a continuous wave. Higher power densities may be employed if pulsed wave ultrasound is used.
  • ultrasound is advantageous as, like light, it may be focused accurately on a target. Moreover, ultrasound is advantageous as it may be focused more deeply into tissues unlike light. It is therefore better suited to whole-tissue penetration (such as but not limited to a lobe of the liver) or whole organ (such as but not limited to the entire liver or an entire muscle, such as the heart) therapy. Another important advantage is that ultrasound is a non-invasive stimulus which is used in a wide variety of diagnostic and therapeutic applications. By way of example, ultrasound is well known in medical imaging techniques and, additionally, in orthopedic therapy. Furthermore, instruments suitable for the application of ultrasound to a subject vertebrate are widely available and their use is well known in the art.
  • the rapid transcriptional response and endogenous targeting of the instant invention make for an ideal system for the study of transcriptional dynamics.
  • the instant invention may be used to study the dynamics of variant production upon induced expression of a target gene.
  • mR A degradation studies are often performed in response to a strong extracellular stimulus, causing expression level changes in a plethora of genes.
  • the instant invention may be utilized to reversibly induce transcription of an endogenous target, after which point stimulation may be stopped and the degradation kinetics of the unique target may be tracked.
  • the temporal precision of the instant invention may provide the power to time genetic regulation in concert with experimental interventions.
  • targets with suspected involvement in long-term potentiation may be modulated in organotypic or dissociated neuronal cultures, but only during stimulus to induce LTP, so as to avoid interfering with the normal development of the cells.
  • LTP long-term potentiation
  • targets suspected to be involved in the effectiveness of a particular therapy may be modulated only during treatment.
  • genetic targets may be modulated only during a pathological stimulus. Any number of experiments in which timing of genetic cues to external experimental stimuli is of relevance may potentially benefit from the utility of the instant invention.
  • the in vivo context offers equally rich opportunities for the instant invention to control gene expression.
  • Photoinducibility provides the potential for spatial precision.
  • a stimulating fiber optic lead may be placed in a precise brain region. Stimulation region size may then be tuned by light intensity. This may be done in conjunction with the delivery of the Casl3 CRISPR-Cas system or complex of the invention, or, in the case of transgenic Casl3 animals, guide RNA of the invention may be delivered and the optrode technology can allow for the modulation of gene expression in precise brain regions.
  • a transparent Casl3 expressing organism can have guide RNA of the invention administered to it and then there can be extremely precise laser induced local gene expression changes.
  • a culture medium for culturing host cells includes a medium commonly used for tissue culture, such as M199-earle base, Eagle MEM (E-MEM), Dulbecco MEM (DMEM), SC-UCM102, UP-SFM (GIBCO BRL), EX-CELL302 (Nichirei), EX-CELL293-S (Nichirei), TFBM-01 (Nichirei), ASF104, among others.
  • Suitable culture media for specific cell types may be found at the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC).
  • Culture media may be supplemented with amino acids such as L-glutamine, salts, anti-fungal or anti-bacterial agents such as Fungizone®, penicillin-streptomycin, animal serum, and the like.
  • the cell culture medium may optionally be serum -free.
  • the invention may also offer valuable temporal precision in vivo.
  • the invention may be used to alter gene expression during a particular stage of development.
  • the invention may be used to time a genetic cue to a particular experimental window.
  • genes implicated in learning may be overexpressed or repressed only during the learning stimulus in a precise region of the intact rodent or primate brain.
  • the invention may be used to induce gene expression changes only during particular stages of disease development. For example, an oncogene may be overexpressed only once a tumor reaches a particular size or metastatic stage.
  • proteins suspected in the development of Alzheimer's may be knocked down only at defined time points in the animal's life and within a particular brain region.
  • Enzymes according to the invention can be used in combination with protected guide RNAs
  • an object of the current invention is to further enhance the specificity of Casl3 given individual guide RNAs through thermodynamic tuning of the binding specificity of the guide RNA to target DNA.
  • This is a general approach of introducing mismatches, elongation or truncation of the guide sequence to increase / decrease the number of complimentary bases vs. mismatched bases shared between a genomic target and its potential off-target loci, in order to give thermodynamic advantage to targeted genomic loci over genomic off-targets.
  • the invention provides for the guide sequence being modified by secondary structure to increase the specificity of the Casl3 CRISPR-Cas system and whereby the secondary structure can protect against exonuclease activity and allow for 3' additions to the guide sequence.
  • the invention provides for hybridizing a "protector RNA” to a guide sequence, wherein the "protector RNA” is an RNA strand complementary to the 5 ' end of the guide RNA (gRNA), to thereby generate a partially double-stranded gRNA.
  • protecting the mismatched bases with a perfectly complementary protector sequence decreases the likelihood of target DNA binding to the mismatched base pairs at the 3' end.
  • additional sequences comprising an extended length may also be present.
  • gRNA Guide RNA extensions matching the genomic target provide gRNA protection and enhance specificity. Extension of the gRNA with matching sequence distal to the end of the spacer seed for individual genomic targets is envisaged to provide enhanced specificity. Matching gRNA extensions that enhance specificity have been observed in cells without truncation. Prediction of gRNA structure accompanying these stable length extensions has shown that stable forms arise from protective states, where the extension forms a closed loop with the gRNA seed due to complimentary sequences in the spacer extension and the spacer seed. These results demonstrate that the protected guide concept also includes sequences matching the genomic target sequence distal of the 20mer spacer-binding region. Thermodynamic prediction can be used to predict completely matching or partially matching guide extensions that result in protected gRNA states.
  • X will generally be of length 17-20nt and Z is of length l-30nt.
  • Thermodynamic prediction can be used to determine the optimal extension state for Z, potentially introducing small numbers of mismatches in Z to promote the formation of protected conformations between X and Z.
  • X and seed length are used interchangeably with the term exposed length (EpL) which denotes the number of nucleotides available for target DNA to bind;
  • EpL exposed length
  • PL protector length
  • Z Z
  • E E
  • ⁇ ' extended length
  • EL extended length
  • An extension sequence which corresponds to the extended length may optionally be attached directly to the guide sequence at the 3' end of the protected guide sequence.
  • the extension sequence may be 2 to 12 nucleotides in length.
  • ExL may be denoted as 0, 2, 4, 6, 8, 10 or 12 nucleotides in length..
  • the ExL is denoted as 0 or 4 nucleotides in length.
  • the ExL is 4 nucleotides in length.
  • the extension sequence may or may not be complementary to the target sequence.
  • An extension sequence may further optionally be attached directly to the guide sequence at the 5' end of the protected guide sequence as well as to the 3' end of a protecting sequence.
  • the extension sequence serves as a linking sequence between the protected sequence and the protecting sequence. Without wishing to be bound by theory, such a link may position the protecting sequence near the protected sequence for improved binding of the protecting sequence to the protected sequence.
  • the invention provides for hybridizing a "protector RNA” to a guide sequence, wherein the "protector RNA” is an RNA strand complementary to the 3 ' end of the guide RNA (gRNA), to thereby generate a partially double-stranded gRNA.
  • gRNA guide RNA
  • the invention provides for enhanced Cas 13 specificity wherein the double stranded 3' end of the protected guide RNA (pgRNA) allows for two possible outcomes: (1) the guide RNA-protector RNA to guide RNA -target DNA strand exchange will occur and the guide will fully bind the target, or (2) the guide RNA will fail to fully bind the target and because Cas 13 target cleavage is a multiple step kinetic reaction that requires guide RNA:target RNA binding to activate Casl3Casl3- catalyzed DSBs, wherein Cas 13 cleavage does not occur if the guide RNA does not properly bind.
  • pgRNA protected guide RNA
  • the protected guide RNA improves specificity of target binding as compared to a naturally occurring CRISPR-Cas system.
  • the protected modified guide RNA improves stability as compared to a naturally occurring CRISPR-Cas.
  • the protector sequence has a length between 3 and 120 nucleotides and comprises 3 or more contiguous nucleotides complementary to another sequence of guide or protector.
  • the protector sequence forms a hairpin.
  • the guide RNA further comprises a protected sequence and an exposed sequence.
  • the exposed sequence is 1 to 19 nucleotides. More particularly, the exposed sequence is at least 75%, at least 90% or about 100% complementary to the target sequence.
  • the guide sequence is at least 90% or about 100% complementary to the protector strand. According to particular embodiments the guide sequence is at least 75%, at least 90% or about 100% complementary to the target sequence.
  • the guide RNA further comprises an extension sequence. More particularly, when the distal end of the guide is the 3' end, the extension sequence is operably linked to the 3 ' end of the protected guide sequence, and optionally directly linked to the 3' end of the protected guide sequence. According to particular embodiments the extension sequence is 1-12 nucleotides.
  • the extension sequence is operably linked to the guide sequence at the 3 ' end of the protected guide sequence and the 5 ' end of the protector strand and optionally directly linked to the 3 ' end of the protected guide sequence and the 5 ' end of the protector strand, wherein the extension sequence is a linking sequence between the protected sequence and the protector strand.
  • the extension sequence is 100% not complementary to the protector strand, optionally at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% not complementary to the protector strand.
  • the guide sequence further comprises mismatches appended to the end of the guide sequence, wherein the mismatches thermodynamically optimize specificity.
  • guide modifications that impede strand invasion will be desireable.
  • it will be desireable to design or modify a guide to impede strand invasiom at off-target sites.
  • it may be acceptable or useful to design or modify a guide at the expense of on-target binding efficiency.
  • guide-target mismatches at the target site may be tolerated that substantially reduce off-target activity.
  • thermodynamic prediction algoithms are used to predict strengths of binding on target and off target.
  • selection methods are used to reduce or minimize off-target effects, by absolute measures or relative to on- target effects.
  • Design options include, without limitation, i) adjusting the length of protector strand that binds to the protected strand, ii) adjusting the length of the portion of the protected strand that is exposed, iii) extending the protected strand with a stem-loop located external (distal) to the protected strand (i.e.
  • the stem loop is external to the protected strand at the distal end
  • iv extending the protected strand by addition of a protector strand to form a stem -loop with all or part of the protected strand
  • addition of a non-structured protector to the end of the protected strand.
  • the invention provides an engineered, non- naturally occurring CRISPR-Cas system comprising a Cas 13 protein and a protected guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the protected guide RNA targets the DNA molecule encoding the gene product and the Cas 13 protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas 13 protein and the protected guide RNA do not naturally occur together.
  • the invention comprehends the protected guide RNA comprising a guide sequence fused to a direct repeat sequence.
  • the invention further comprehends the Cas 13 CRISPR protein being codon optimized for expression in a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell and in a more preferred embodiment the mammalian cell is a human cell.
  • the expression of the gene product is decreased.
  • the CRISPR protein is Cas 13.
  • the CRISPR protein is Casl2a.
  • the Casl3Casl2a protein is Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium or Francisella Novicida Casl3Casl2a, and may include mutated Cas 12a derived from these organisms.
  • the protein may be a further Cas9 or Cas 12a homolog or ortholog.
  • the nucleotide sequence encoding the Csa9 or Cas 12a protein is codon-optimized for expression in a eukaryotic cell.
  • the Cas 13 or Cas 12a protein directs cleavage of one or two strands at the location of the target sequence.
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively- linked. Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.
  • the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with the guide RNA comprising the guide sequence that is hybridized to the target sequence and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Casl3 enzyme comprising a nuclear localization sequence.
  • the host cell comprises components (a) and (b).
  • component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell.
  • the Casl3 enzyme directs cleavage of one or two strands at the location of the target sequence.
  • the Casl3 enzyme lacks DNA strand cleavage activity.
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments.
  • the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments.
  • the organism in some embodiments of these aspects may be an animal; for example a mammal. Also, the organism may be an arthropod such as an insect. The organism also may be a plant or a yeast. Further, the organism may be a fungus.
  • the invention provides a kit comprising one or more of the components described herein above.
  • the kit comprises a vector system and instructions for using the kit.
  • the vector system comprises (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Casl3 CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a Casl3 enzyme complexed with the protected guide RNA comprising the guide sequence that is hybridized to the target sequence and/or (b) a second regulatory element operably linked to an enzyme- coding sequence encoding said Casl3 enzyme comprising a nuclear localization sequence.
  • the kit comprises components (a) and (b) located on the same or different vectors of the system.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell.
  • the Casl3 enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said Casl3 enzyme in a detectable amount in the nucleus of a eukaryotic cell. .
  • the enzyme may be a Cas9 homolog or ortholog.
  • the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.
  • the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter.
  • the invention provides a method of modifying a target polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a Casl3 enzyme complexed with protected guide RNA comprising a guide sequence hybridized to a target sequence within said target polynucleotide.
  • said cleavage comprises cleaving one or two strands at the location of the target sequence by said Casl3 enzyme.
  • said cleavage results in decreased transcription of a target gene.
  • the method further comprises repairing said cleaved target polynucleotide by non-homologous end joining (NHEJ)-based gene insertion mechanisms, more particularly with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.
  • said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the Casl3 enzyme, the protected guide RNA comprising the guide sequence linked to direct repeat sequence.
  • said vectors are delivered to the eukaryotic cell in a subject.
  • said modifying takes place in said eukaryotic cell in a cell culture.
  • the method further comprises isolating said eukaryotic cell from a subject prior to said modifying.
  • the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a Casl3 CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a Casl3 enzyme complexed with a protected guide RNA comprising a guide sequence hybridized to a target sequence within said polynucleotide.
  • the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the Casl3 enzyme and the protected guide RNA.
  • the invention provides a method of generating a model eukaryotic cell comprising a mutated disease gene.
  • a disease gene is any gene associated an increase in the risk of having or developing a disease.
  • the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive expression of one or more of: a Casl3 enzyme and a protected guide RNA comprising a guide sequence linked to a direct repeat sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said disease gene, wherein the CRISPR complex comprises the Cas 13 enzyme complexed with the guide RNA comprising the sequence that is hybridized to the target sequence within the target polynucleotide, thereby generating a model eukaryotic cell comprising a mutated disease gene.
  • said cleavage comprises cleaving one or two strands at the location of the target sequence by said Cas 13 enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by non-homologous end joining (NHEI)-based gene insertion mechanisms with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expression from a gene comprising the target sequence.
  • NHEI non-homologous end joining
  • the invention provides a method for developing a biologically active agent that modulates a cell signaling event associated with a disease gene.
  • a disease gene is any gene associated an increase in the risk of having or developing a disease.
  • the method comprises (a) contacting a test compound with a model cell of any one of the described embodiments; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with said mutation in said disease gene, thereby developing said biologically active agent that modulates said cell signaling event associated with said disease gene.
  • the invention provides a recombinant polynucleotide comprising a protected guide sequence downstream of a direct repeat sequence, wherein the protected guide sequence when expressed directs sequence-specific binding of a CRISPR complex to a corresponding target sequence present in a eukaryotic cell.
  • the target sequence is a viral sequence present in a eukaryotic cell.
  • the target sequence is a proto-oncogene or an oncogene.
  • the invention provides for a method of selecting one or more cell(s) by introducing one or more mutations in a gene in the one or more cell (s), the method comprising: introducing one or more vectors into the cell (s), wherein the one or more vectors drive expression of one or more of: a Cas 13 enzyme, a protected guide RNA comprising a guide sequence, and an editing template; wherein the editing template comprises the one or more mutations that abolish Cas 13 enzyme cleavage; allowing nonhomologous end joining (NHEJ)-based gene insertion mechanisms of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises the Cas 13 enzyme complexed with the protected guide RNA comprising a guide sequence that is hybridized to the target sequence within the target polynu
  • NHEJ nonhom
  • the invention provides as to any or each or all embodiments herein-discussed wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutation or the at least two or more mutations are selected from those described herein elsewhere.
  • the invention involves a computer-assisted method for identifying or designing potential compounds to fit within or bind to CRISPR-Cas 13 system or a functional portion thereof or vice versa (a computer-assisted method for identifying or designing potential CRISPR-Cas 13 systems or a functional portion thereof for binding to desired compounds) or a computer-assisted method for identifying or designing potential CRISPR-Casl3 systems (e.g., with regard to predicting areas of the CRISPR-Cas 13 system to be able to be manipulated— for instance, based on crystal structure data or based on data of Cas 13 orthologs, or with respect to where a functional group such as an activator or repressor can be attached to the CRISPR-Cas 13 system, or as to Cas 13 truncations or as to designing nickases), said method comprising:
  • a computer system e.g., a programmed computer comprising a processor, a data storage system, an input device, and an output device, the steps of:
  • said method comprising: providing the co-ordinates of at least two atoms of the CRISPR- Casl3 crystal structure, e.g., at least two atoms of the herein Crystal Structure Table of the CRISPR-Casl3 crystal structure or co-ordinates of at least a sub-domain of the CRISPR-Casl3 crystal structure ("selected co-ordinates"), providing the structure of a candidate comprising a binding molecule or of portions of the CRISPR-Casl3 system that may be manipulated, e.g., based on data from other portions of the CRISPR- Casl3 crystal structure and/or from Casl3 orthologs, or the structure of functional groups, and fitting the structure of the candidate to the selected co-ordinates, to thereby obtain product data comprising CRISPR- Casl3 structures that may bind to desired structures, desired structures that may bind to certain CRISPR- Casl3 structures, portions of the CRISPR-Casl3 system
  • the testing can comprise analyzing the CRISPR-Casl3 system resulting from said synthesized selected structure(s), e.g., with respect to binding, or performing a desired function.
  • the output in the foregoing methods can comprise data transmission, e.g., transmission of information via telecommunication, telephone, video conference, mass communication, e.g., presentation such as a computer presentation (e.g. POWERPOINT), internet, email, documentary communication such as a computer program (e.g. WORD) document and the like.
  • the invention also comprehends computer readable media containing: atomic co-ordinate data according to the herein-referenced Crystal Structure, said data defining the three dimensional structure of CRISPR-Casl3 or at least one sub-domain thereof, or structure factor data for CRISPR-Casl3, said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure.
  • the computer readable media can also contain any data of the foregoing methods.
  • the invention further comprehends methods a computer system for generating or performing rational design as in the foregoing methods containing either: atomic coordinate data according to herein-referenced Crystal Structure, said data defining the three dimensional structure of CRISPR-Casl3 or at least one sub-domain thereof, or structure factor data for CRISPR-Casl3, said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure.
  • the invention further comprehends a method of doing business comprising providing to a user the computer system or the media or the three dimensional structure of CRISPR-Casl3 or at least one sub- domain thereof, or structure factor data for CRISPR-Casl3, said structure set forth in and said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure, or the herein computer media or a herein data transmission.
  • a "binding site” or an “active site” comprises or consists essentially of or consists of a site (such as an atom, a functional group of an amino acid residue or a plurality of such atoms and/or groups) in a binding cavity or region, which may bind to a compound such as a nucleic acid molecule, which is/are involved in binding.
  • fitting is meant determining by automatic, or semi-automatic means, interactions between one or more atoms of a candidate molecule and at least one atom of a structure of the invention, and calculating the extent to which such interactions are stable. Interactions include attraction and repulsion, brought about by charge, steric considerations and the like. Various computer-based methods for fitting are described further
  • root mean square (or rms) deviation we mean the square root of the arithmetic mean of the squares of the deviations from the mean.
  • a “computer system” By a “computer system”, is meant the hardware means, software means and data storage means used to analyze atomic coordinate data.
  • the minimum hardware means of the computer-based systems of the present invention typically comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a display or monitor is provided to visualize structure data.
  • the data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are computer and tablet devices running Unix, Windows or Apple operating systems.
  • computer readable media any medium or media, which can be read and accessed directly or indirectly by a computer e.g., so that the media is suitable for use in the above-mentioned computer system.
  • Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; thumb drive devices; cloud storage devices and hybrids of these categories such as magnetic/optical storage media.
  • the invention comprehends the use of the protected guides described herein above in the optimized functional CRISPR-Cas enzyme systems described herein.
  • the nucleic acid molecule encoding a Cas is advantageously codon optimized Cas.
  • An example of a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • an enzyme coding sequence encoding a Cas is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a Cas correspond to the most frequently used codon for a particular amino acid.
  • the methods as described herein may comprise providing a Cas transgenic cell in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced operably connected in the cell with a regulatory element comprising a promoter of one or more gene of interest.
  • a Cas transgenic cell refers to a cell, such as a eukaryotic cell, in which a Cas gene has been genomically integrated. The nature, type, or origin of the cell are not particularly limiting according to the present invention. Also the way how the Cas transgene is introduced in the cell is may vary and can be any method as is known in the art.
  • the Cas transgenic cell is obtained by introducing the Cas transgene in an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism.
  • the Cas transgenic cell as referred to herein may be derived from a Cas transgenic eukaryote, such as a Cas knock-in eukaryote.
  • WO 2014/093622 PCT/US 13/74667
  • directed to targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention.
  • Methods of US Patent Publication No. 20130236946 assigned to Cellectis directed to targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention.
  • Piatt et. al. Cell; 159(2):440-455 (2014)
  • the Cas transgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas expression inducible by Cre recombinase.
  • the Cas transgenic cell may be obtained by introducing the Cas transgene in an isolated cell. Delivery systems for transgenes are well known in the art.
  • the Cas transgene may be delivered in for instance eukaryotic cell by means of vector (e.g., AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, as also described herein elsewhere.
  • the cell such as the Cas transgenic cell, as referred to herein may comprise further genomic alterations besides having an integrated Cas gene or the mutations arising from the sequence specific action of Cas when complexed with RNA capable of guiding Cas to a target locus, such as for instance one or more oncogenic mutations, as for instance and without limitation described in Piatt et al. (2014), Chen et al., (2014) or Kumar et al.. (2009).
  • the Cas sequence is fused to one or more nuclear localization sequences (NLSs) or nuclear export signals (NESs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs.
  • the Cas comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near the carboxy- terminus, or a combination of these (e.g. zero or at least one or more NLS or NES at the amino- terminus and zero or at one or more NLS or NES at the carboxy terminus).
  • the Cas comprises at most 6 NLSs.
  • an NLS or NES is considered near the N- or C- terminus when the nearest amino acid of the NLS or NES is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • Non- limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV(SEQ ID NO: 1); the NLS from nucleoplasmin (e.g.
  • nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK) (SEQ ID NO:2); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO:4); the hRNPAl M9 NLS having the sequence NQS SNFGPMKGGNFGGRS SGP YGGGGQYF AKPRNQGGY(SEQ ID NO: 5); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence POPKKKPL (SEQ ID No: 9) of human p53; the sequence SALIKKKKKMAP (SEQ ID No: 10) of mouse c-abl IV; the
  • NESs include an NES sequence LYPERLRRILT (SEQ ID No.
  • the one or more NLSs or NESs are of sufficient strength to drive accumulation of the Cas in a detectable amount in respectively the nucleus or the cytoplasm of a eukaryotic cell.
  • strength of nuclear localization/export activity may derive from the number of NLSs/NESs in the Cas, the particular NLS(s) or NES(s) used, or a combination of these factors. Detection of accumulation in the nucleus/cytoplasm may be performed by any suitable technique.
  • a detectable marker may be fused to the Cas, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI) or cytoplasm.
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g.
  • RNA cleavage or mutation at the target sequence or assay for altered gene expression activity affected by CRISPR complex formation and/or Cas enzyme activity), as compared to a control no exposed to the Cas or complex, or exposed to a Cas lacking the one or more NLSs or NESs.
  • other localization tags may be fused to the Cas protein, such as without limitation for localizing the Cas to particular sites in a cell, such as organells, such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear or cellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles, centrosome, nucleosome, granules, centrioles, etc.
  • organells such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear or cellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles, centrosome, nucleosome, granules, centrioles, etc.
  • the invention involves vectors, e.g. for delivering or introducing in a cell Cas and/or RNA capable of guiding Cas to a target locus (i.e. guide RNA), but also for propagating these components (e.g. in prokaryotic cells).
  • a "vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the vector(s) can include the regulatory element(s), e.g., promoter(s).
  • the vector(s) can comprise Cas encoding sequences, and/or a single, but possibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s) (e.g., sgRNAs).
  • guide RNA(s) e.g., sgRNAs
  • a promoter for each RNA there can be a promoter for each RNA (e.g., sgRNA), advantageously when there are up to about 16 RNA(s) ; and, when a single vector provides for more than 16 RNA(s) , one or more promoter(s) can drive expression of more than one of the RNA(s), e.g., when there are 32 RNA(s) , each promoter can drive expression of two RNA(s) , and when there are 48 RNA(s) , each promoter can drive expression of three RNA(s) .
  • sgRNA e.g., sgRNA
  • RNA(s) for a suitable exemplary vector such as AAV, and a suitable promoter such as the U6 promoter.
  • a suitable exemplary vector such as AAV
  • a suitable promoter such as the U6 promoter.
  • the packaging limit of AAV is -4.7 kb.
  • the length of a single U6- gRNA (plus restriction sites for cloning) is 361 bp. Therefore, the skilled person can readily fit about 12-16, e.g., 13 U6-gRNA cassettes in a single vector.
  • This can be assembled by any suitable means, such as a golden gate strategy used for TALE assembly (http://www.genome- engineering.org/taleffectors/).
  • the skilled person can also use a tandem guide strategy to increase the number of U6-gRNAs by approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 to approximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled in the art can readily reach approximately 18-24, e.g., about 19 promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector.
  • a further means for 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.
  • AAV may package U6 tandem gRNA targeting up to about 50 genes.
  • vector(s) e.g., a single vector, expressing multiple RNAs or guides under the control or operatively or functionally linked to one or more promoters— especially as to the numbers of RNAs or guides discussed herein, without any undue experimentation.
  • the guide RNA(s) encoding sequences and/or Cas encoding sequences can be functionally or operatively linked to regulatory element(s) and hence the regulatory element(s) drive expression.
  • the promoter(s) can be constitutive promoter(s) and/or conditional promoter(s) and/or inducible promoter(s) and/or tissue specific promoter(s).
  • the promoter can be selected from the group consisting of RNA polymerases, pol I, pol II, pol III, T7, U6, HI, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter the dihydrofolate reductase promoter
  • ⁇ -actin promoter the phosphoglycerol kinase (PGK) promoter
  • PGK phosphoglycerol kinase
  • aspects of the invention relate to the identification and engineering of novel effector proteins associated with Class 2 CRISPR-Cas systems.
  • the effector protein comprises a single-subunit effector module.
  • the effector protein is functional in prokaryotic or eukaryotic cells for in vitro, in vivo or ex vivo applications.
  • An aspect of the invention encompasses computational methods and algorithms to predict new Class 2 CRISPR-Cas systems and identify the components therein.
  • a computational method of identifying novel Class 2 CRISPR-Cas loci comprises the following steps: detecting all contigs encoding the Casl protein; identifying all predicted protein coding genes within 20kB of the casl gene, more particularly within the region 20 kb from the start of the casl gene and 20 kb from the end of the casl gene; comparing the identified genes with Cas protein-specific profiles and predicting CRISPR arrays; selecting partial and/or unclassified candidate CRISPR-Cas loci containing proteins larger than 500 amino acids (>500 aa); analyzing selected candidates using PSI-BLAST and HHPred, thereby isolating and identifying novel Class 2 CRISPR-Cas loci.
  • additional analysis of the candidates may be conducted by searching metagenomics databases for additional homologs.
  • the detecting all contigs encoding the Casl protein is performed by GenemarkS which a gene prediction program as further described in "GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions.” John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, herein incorporated by reference.
  • the identifying all predicted protein coding genes is carried out by comparing the identified genes with Cas protein-specific profiles and annotating them according to NCBI conserveed Domain Database (CDD) which is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST.
  • CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence/structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).
  • CRISPR arrays were predicted using a PILER-CR program which is a public domain software for finding CRISPR repeats as described in "PILER-CR: fast and accurate identification of CRISPR repeats", Edgar, R.C., BMC Bioinformatics, Jan 20; 8: 18(2007), herein incorporated by reference.
  • PSI-BLAST Position- Specific Iterative Basic Local Alignment Search Tool
  • PSSM position-specific scoring matrix
  • PSSM position-specific scoring matrix
  • the case by case analysis is performed using HHpred, a method for sequence database searching and structure prediction that is as easy to use as BLAST or PSI- BLAST and that is at the same time much more sensitive in finding remote homologs.
  • HHpred's sensitivity is competitive with the most powerful servers for structure prediction currently available.
  • HHpred is the first server that is based on the pairwise comparison of profile hidden Markov models (HMMs).
  • HMMs profile hidden Markov models
  • most conventional sequence search methods search sequence databases such as UniProt or the R
  • HHpred searches alignment databases, like Pfam or SMART. This greatly simplifies the list of hits to a number of sequence families instead of a clutter of single sequences.
  • HHpred accepts a single query sequence or a multiple alignment as input. Within only a few minutes it returns the search results in an easy-to-read format similar to that of PSI-BLAST. Search options include local or global alignment and scoring secondary structure similarity. HHpred can produce pairwise query-template sequence alignments, merged query-template multiple alignments (e.g. for transitive searches), as well as 3D structural models calculated by the MODELLER software from HHpred alignments.
  • nucleic acid-targeting system wherein nucleic acid is DNA or RNA, and in some aspects may also refer to DNA-RNA hybrids or derivatives thereof, refers collectively to transcripts and other elements involved in the expression of or directing the activity of DNA or RNA-targeting CRISPR-associated (“Cas") genes, which may include sequences encoding a DNA or RNA-targeting Cas protein and a DNA or RNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (in some but not all systems) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence, or other sequences and transcripts from a DNA or RNA-targeting CRISPR locus.
  • Cas CRISPR-associated
  • a RNA-targeting system is characterized by elements that promote the formation of a DNA or RNA-targeting complex at the site of a target DNA or RNA sequence.
  • target sequence refers to a DNA or RNA sequence to which a DNA or RNA-targeting guide RNA is designed to have complementarity, where hybridization between a target sequence and a RNA-targeting guide RNA promotes the formation of a RNA-targeting complex.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • novel RNA targeting systems also referred to as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas system RNA-targeting system of the present application are based on identified Type VI Cas proteins which do not require the generation of customized proteins to target specific RNA sequences but rather a single enzyme can be programmed by a RNA molecule to recognize a specific RNA target, in other words the enzyme can be recruited to a specific RNA target using said RNA molecule.
  • novel DNA targeting systems also referred to as DNA- or DNA-targeting CRISPR/Cas or the CRISPR-Cas system RNA-targeting system of the present application are based on identified Type VI Cas proteins which do not require the generation of customized proteins to target specific RNA sequences but rather a single enzyme can be programmed by a RNA molecule to recognize a specific DNA target, in other words the enzyme can be recruited to a specific DNA target using said RNA molecule.
  • nucleic acids-targeting systems may be used in various nucleic acids-targeting applications, altering or modifying synthesis of a gene product, such as a protein, nucleic acids cleavage, nucleic acids editing, nucleic acids splicing; trafficking of target nucleic acids, tracing of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, etc.
  • a Cas protein or a CRISPR enzyme refers to any of the proteins presented in the new classification of CRISPR-Cas systems.
  • the Class 2 type VI effector protein Casl3c is a RNA-guided RNase that can be efficiently programmed to degrade ssRNA.
  • Cas 13c effector proteins of the invention include, without limitation, the following orthlog species (including multiple CRISPR loci): Fusobacterium necrophorum subsp. funduliforme ATCC 51357; Fusobacterium necrophorum DJ-2; Fusobacterium necrophorum BFTR-1; Fusobacterium necrophorum subsp.
  • Casl3c achieves RNA cleavage through conserved basic residues within its two HEPN domains. Mutation of the HEPN domain, such as (e.g. alanine) substitution of predicted HEPN domain catalytic residues can be used to convert Casl3c into an inactive programmable RNA- binding protein (dCasl3c, analogous to dCas9).
  • dCasl3c inactive programmable RNA- binding protein
  • a consensus sequence can be generated from multiple C2c2 orthologs, which can assist in locating conserved amino acid residues, and motifs, including but not limited to catalytic residues and HEPN motifs in Casl3c orthologs that mediate Casl3c function.
  • One such consensus sequence generated from the 33 orthologs mentioned above using
  • HEPN sequence motifs identified from the above orthologs are provided in Figs. 49 and 50 on the basis of the first 21 orthologs and all 33 orthologs respectively.
  • Non-limiting examples of amino acid residues that can be mutated to generate catalytically dead C2c2 mutants, based on the above consensus include, in or near HEPNl, D372, R377, Q/H382, and F383 or corresponding amino acids of an ortholog, and in or near HEPN2, K893, N894, R898, N899, H903, F904, Y906, Y927, D928, K930, K932 or corresponding amino acids of an ortholog.
  • a sequence alignment tool to assist generation of a consensus sequence and identification of conserved residues is the MUSCLE alignment tool (www. e i . ac. uk Fools/msa musci e ).
  • Casl3c HEPN may also target DNA, or potentially DNA and/or RNA.
  • the HEPN domains of Casl3c are at least capable of binding to and, in their wild-type form, cutting RNA, then it is preferred that the Casl3c effector protein has RNase function. It may also, or alternatively, have DNase function.
  • the effector protein may be a RNA-binding protein, such as a dead-Cas type effector protein, which may be optionally functionalized as described herein for instance with an transcriptional activator or repressor domain, NLS or other functional domain.
  • the effector protein may be a RNA-binding protein that cleaves a single strand of RNA. If the RNA bound is ssRNA, then the ssRNA is fully cleaved.
  • the effector protein may be a RNA-binding protein that cleaves a double strand of RNA, for example if it comprises two RNase domains. If the RNA bound is dsRNA, then the dsRNA is fully cleaved.
  • RNase function in CRISPR systems is known, 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) and provides significant advantages.
  • the target RNA i.e. the RNA of interest
  • the target RNA is the RNA to be targeted by the present invention leading to the recruitment to, and the binding of the effector protein at, the target site of interest on the target RNA.
  • the target RNA may be any suitable form of RNA. This may include, in some embodiments, mRNA. In other embodiments, the target RNA may include tRNA or rRNA. In other embodiments, the target RNA may include miRNA. In other embodiments, the target RNA may include siRNA.
  • the effecteor protein (CRISPR enzyme; Casl3; effector protein) according to the invention as described herein is a catalytically inactive or dead Casl3 effector protein (dCasl3).
  • the dCasl3 effector comprises mutations in the nuclease domain.
  • the dCasl3 effector protein has been truncated. To reduce the size of a fusion protein of the Casl3 effector and the one or more functional domains, the C- terminus of the Casl3 effector can be truncated while still maintaining its RNA binding function.
  • Casl3 truncations include C-terminal ⁇ 984-1090, C-terminal ⁇ 1026-1090, and C-terminal ⁇ 1053-1090, C-terminal ⁇ 934-1090, C-terminal ⁇ 884-1090, C-terminal ⁇ 834-1090, C-terminal ⁇ 784-1090, and C-terminal ⁇ 734-1090, wherein amino acid positions correspond to amino acid positions of Prevotella sp. P5-125 Casl3b protein. See Fig. 4.
  • RNAi Interfering RNA
  • miRNA microRNA
  • the target RNA may include interfering RNA, i.e. RNA involved in an RNA interference pathway, such as shRNA, siRNA and so forth, both in eukaryotes and prokaryotes.
  • the target RNA may include microRNA (miRNA). Control over interfering RNA or miRNA may help reduce off-target effects (OTE) seen with those approaches by reducing the longevity of the interfering RNA or miRNA in vivo or in vitro.
  • the target is not the miRNA itself, but the miRNA binding site of a miRNA target.
  • miRNAs may be sequestered (such as including subcellularly relocated). In certain embodiments, miRNAs may be cut, such as without limitation at hairpins.
  • miRNA processing (such as including turnover) is increased or decreased.
  • the effector protein and suitable guide are selectively expressed (for example spatially or temporally under the control of a suitable promoter, for example a tissue- or cell cycle- specific promoter and/or enhancer) then this could be used to 'protect' the cells or systems (in vivo or in vitro) from RNAi in those cells.
  • a suitable promoter for example a tissue- or cell cycle- specific promoter and/or enhancer
  • This may be useful in neighbouring tissues or cells where RNAi is not required or for the purposes of comparison of the cells or tissues where the effector protein and suitable guide are and are not expressed (i.e. where the RNAi is not controlled and where it is, respectively).
  • the effector protein may be used to control or bind to molecules comprising or consisting of RNA, such as ribozymes, ribosomes or riboswitches.
  • the RNA guide can recruit the effector protein to these molecules so that the effector protein is able to bind to them.
  • the protein system of the invention can be applied in areas of RNAi technologies, without undue experimentation, from this disclosure, including therapeutic, assay and other applications (see, e.g., Guidi et al., PLoS Negl Trop Dis 9(5): e0003801. doi: 10.1371/journal.pntd; Crotty et al., In vivo RNAi screens: concepts and applications. Shane Crotty ... 2015 Elsevier Ltd. Published by Elsevier Inc., Pesticide Biochemistry and Physiology (Impact Factor: 2.01). 01/2015; 120.
  • rRNA Ribosomal RNA
  • azalide antibiotics such as azithromycin
  • the present effector protein together with a suitable guide RNA to target the 50S ribosomal subunit, may be, in some embodiments, recruited to and bind to the 50S ribosomal subunit.
  • the present effector protein in concert with a suitable guide directed at a ribosomal (especially the 50s ribosomal subunit) target is provided.
  • Use of this use effector protein in concert with the suitable guide directed at the ribosomal (especially the 50s ribosomal subunit) target may include antibiotic use.
  • the antibiotic use is analogous to the action of azalide antibiotics, such as azithromycin.
  • prokaryotic ribosomal subunits such as the 70S subunit in prokaryotes, the 50S subunit mentioned above, the 30S subunit, as well as the 16S and 5S subunits may be targeted.
  • eukaryotic ribosomal subunits such as the 80S subunit in eukaryotes, the 60S subunit, the 40S subunit, as well as the 28S, 18S. 5.8S and 5S subunits may be targeted.
  • the effector protein may be a RNA-binding protein, optionally functionalized, as described herein.
  • the effector protein may be a RNA- binding protein that cleaves a single strand of RNA. In either case, but particularly where the RNA-binding protein cleaves a single strand of RNA, then ribosomal function may be modulated and, in particular, reduced or destroyed. This may apply to any ribosomal RNA and any ribosomal subunit and the sequences of rRNA are well known.
  • Control of ribosomal activity is thus envisaged through use of the present effector protein in concert with a suitable guide to the ribosomal target. This may be through cleavage of, or binding to, the ribosome.
  • reduction of ribosomal activity is envisaged. This may be useful in assaying ribosomal function in vivo or in vitro, but also as a means of controlling therapies based on ribosomal activity, in vivo or in vitro.
  • control (i.e. reduction) of protein synthesis in an in vivo or in vitro system is envisaged, such control including antibiotic and research and diagnostic use.
  • a riboswitch (also known as an aptozyme) is a regulatory segment of a messenger RNA molecule that binds a small molecule. This typically results in a change in production of the proteins encoded by the mRNA.
  • control of riboswitch activity is thus envisaged through use of the present effector protein in concert with a suitable guide to the riboswitch target. This may be through cleavage of, or binding to, the riboswitch. In particular, reduction of riboswitch activity is envisaged.
  • Ribozymes may be useful in assaying riboswitch function in vivo or in vitro, but also as a means of controlling therapies based on riboswitch activity, in vivo or in vitro. Furthermore, control (i.e. reduction) of protein synthesis in an in vivo or in vitro system is envisaged.
  • This control as for rRNA may include antibiotic and research and diagnostic use.
  • Ribozymes are RNA molecules having catalytic properties, analogous to enzymes (which are of course proteins). As ribozymes, both naturally occurring and engineered, comprise or consist of RNA, they may also be targeted by the present RNA-binding effector protein. In some embodiments, the effector protein may be a RNA-binding protein cleaves the ribozyme to thereby disable it. Control of ribozymal activity is thus envisaged through use of the present effector protein in concert with a suitable guide to the ribozymal target. This may be through cleavage of, or binding to, the ribozyme. In particular, reduction of ribozymal activity is envisaged. This may be useful in assaying ribozymal function in vivo or in vitro, but also as a means of controlling therapies based on ribozymal activity, in vivo or in vitro.
  • the effector protein may also be used, together with a suitable guide, to target gene expression, including via control of RNA processing.
  • the control of RNA processing may include RNA processing reactions such as RNA splicing, including alternative splicing, via targeting of RNApol; viral replication (in particular of satellite viruses, bacteriophages and retroviruses, such as HBV, HBC and HIV and others listed herein) including virioids in plants; and tRNA biosynthesis.
  • the effector protein and suitable guide may also be used to control RNAactivation (RNAa).
  • RNAa leads to the promotion of gene expression, so control of gene expression may be achieved that way through disruption or reduction of RNAa and thus less promotion of gene expression. This is discussed more in detail below.
  • RNAi screens Identifying gene products whose knockdown is associated with phenotypic changes, biological pathways can be interrogated and the constituent parts identified, via RNAi screens. Control may also be exerted over or during these screens by use of the effector protein and suitable guide to remove or reduce the activity of the RNAi in the screen and thus reinstate the activity of the (previously interfered with) gene product (by removing or reducing the interference/repression).
  • Satellite RNAs and satellite viruses may also be treated.
  • Control herein with reference to RNase activity generally means reduction, negative disruption or known-down or knock out.
  • the target-specific RNAses provided herein allow for very specific cutting of a target RNA.
  • the interference at RNA level allows for modulation both spatially and temporally and in a non-invasive way, as the genome is not modified.
  • mRNA targets examples include VEGF, VEGF- Rl and RTP801 (in the treatment of AMD and/or DME), Caspase 2 (in the treatment of Naion)ADRB2 (in the treatment of intraocular pressure), TRPVI (in the treatment of Dry eye syndrome, Syk kinase (in the treatment of asthma), Apo B (in the treatment of hypercholesterolemia or hypobetalipoproteinemia), PLK1, KSP and VEGF (in the treatment of solid tumors), Ber-Abl (in the treatment of CML)(Burnett and Rossi Chem Biol. 2012, 19(1): 60- 71)).
  • RNA targeting has been demonstrated to be effective in 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).
  • HIV targeting of HIV Tet and Rev
  • RSV targeting of RSV nucleocapsid
  • HCV targeting of miR-122
  • RNA targeting effector protein of the invention can be used for mutation specific or allele specific knockdown.
  • Guide RNA's can be designed that specifically target a sequence in the transcribed mRNA comprising a mutation or an allele-specific sequence.
  • Such specific knockdown is particularly suitable for therapeutic applications relating 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.
  • FHBL familial hypobetalipoproteinemia
  • This gene encodes two versions of the apolipoprotein B protein: a short version (ApoB-48) and a longer version (ApoB-100).
  • RNA targeting effector protein of the invention may be beneficial in treatment of FHBL.
  • Huntington's disease is caused by an expansion of CAG triplet repeats in the gene coding for the Huntingtin protein, which results in an abnormal protein.
  • mutated or allele-specific mRNA transcripts encoding the Huntingtin protein with an RNA targeting effector protein of the invention may be beneficial in treatment of HD.
  • the Casl3c is split in the sense that the two parts of the Casl3c enzyme substantially comprise a functioning Casl3c. Ideally, the split should always be so that the catalytic domain(s) are unaffected. That Casl3c may function as a nuclease or it may be a dead-Casl3c which is essentially an RNA-binding protein with very little or no catalytic activity, due to typically mutation(s) in its catalytic domains.
  • Each half of the split Casl3c may be fused to a dimerization partner.
  • employing rapamycin sensitive dimerization domains allows to generate a chemically inducible split Casl3c for temporal control of Cas 13 cacti vity.
  • Casl3c can thus be rendered chemically inducible by being split into two fragments and that rapamycin- sensitive dimerization domains may be used for controlled reassembly of the Cas 13 c.
  • the two parts of the split Casl3c can be thought of as the N' terminal part and the C terminal part of the split Casl3c.
  • the fusion is typically at the split point of the Casl3c.
  • the C terminal of the N' terminal part of the split Casl3c is fused to one of the dimer halves, whilst the N' terminal of the C terminal part is fused to the other dimer half.
  • the Casl3c does not have to be split in the sense that the break is newly created.
  • the split point is typically designed in silico and cloned into the constructs.
  • the two parts of the split Casl3c, the N' terminal and C terminal parts form a full Casl3c, comprising preferably at least 70% or more of the wildtype amino acids (or nucleotides encoding them), preferably at least 80%) or more, preferably at least 90% or more, preferably at least 95% or more, and most preferably at least 99% or more of the wildtype amino acids (or nucleotides encoding them).
  • Some trimming may be possible, and mutants are envisaged.
  • Non-functional domains may be removed entirely. What is important is that the two parts may be brought together and that the desired Casl3c function is restored or reconstituted.
  • the dimer may be a homodimer or a heterodimer.
  • the Casl3c effector as described herein may be used for mutation-specific, or allele-specific targeting, such as . for mutation-specific, or allele-specific knockdown.
  • RNA targeting effector protein can moreover be fused to another functional RNAse domain, such as a non-specific RNase or Argonaute 2, which acts in synergy to increase the RNAse activity or to ensure further degradation of the message.
  • a non-specific RNase or Argonaute 2 acts in synergy to increase the RNAse activity or to ensure further degradation of the message.
  • RNA targeting can also be used to impact specific aspects of the RNA processing within the cell, which may allow a more subtle modulation of gene expression.
  • modulation can for instance be mediated by interfering with binding of proteins to the RNA, such as for instance blocking binding of proteins, or recruiting RNA binding proteins.
  • modulations can be ensured at different levels such as splicing, transport, localization, translation and turnover of the mRNA.
  • it can be envisaged to address (pathogenic) malfunctioning at each of these levels by using RNA-specific targeting molecules.
  • the RNA targeting protein is a "dead" Casl3c that has lost the ability to cut the RNA target but maintains its ability to bind thereto, such as the mutated forms of Casl3c described herein.
  • RNA targeting effector proteins described herein can for instance be used to block or promote slicing, include or exclude exons and influence the expression of specific isoforms and/or stimulate the expression of alternative protein products. Such applications are described in more detail below.
  • a RNA targeting effector protein binding to a target RNA can sterically block access of splicing factors to the RNA sequence.
  • the RNA targeting effector protein targeted to a splice site may block splicing at the site, optionally redirecting splicing to an adjacent site.
  • a RNA targeting effector protein binding to the 5' splice site binding can block the recruitment of the Ul component of the spliceosome, favoring the skipping of that exon.
  • a RNA targeting effector protein targeted to a splicing enhancer or silencer can prevent binding of transacting regulatory splicing factors at the target site and effectively block or promote splicing.
  • Exon exclusion can further be achieved by recruitment of ILF2/3 to precursor mRNA near an exon by an RNA targeting effector protein as described herein.
  • a glycine rich domain can be attached for recruitment of hnRNP Al and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999 Jan; 19(1):251-60).
  • splice variants may be targeted, while other splice variants will not be targeted.
  • the RNA targeting effector protein can be used to promote slicing (e.g. where splicing is defective).
  • a RNA targeting effector protein can be associated with an effector capable of stabilizing a splicing regulatory stem-loop in order to further splicing.
  • the RNA targeting effector protein can be linked to a consensus binding site sequence for a specific splicing factor in order to recruit the protein to the target DNA.
  • Examples of diseases which have been associated with aberrant splicing include, but are not limited to Paraneoplastic Opsoclonus Myoclonus Ataxia (or POMA), resulting from a loss of Nova proteins which regulate splicing of proteins that function in the synapse, and Cystic Fibrosis, which is caused by defective splicing of a cystic fibrosis transmembrane conductance regulator, resulting in the production of nonfunctional chloride channels.
  • aberrant RNA splicing results in gain-of-function. This is the case for instance in myotonic dystrophy which is caused by a CUG triplet-repeat expansion (from 50 to >1500 repeats) in the 3'UTR of an mRNA, causing splicing defects.
  • the RNA targeting effector protein can be used to include an exon by recruiting a splicing factor (such as Ul) to a 5' splicing site to promote excision of introns around a desired exon. Such recruitment could be mediated trough a fusion with an arginine/serine rich domain, which functions as splicing activator (Gravely BR and Maniatis T, Mol Cell. 1998 (5):765-71). [00403] It is envisaged that the RNA targeting effector protein can be used to block the splicing machinery at a desired locus, resulting in preventing exon recognition and the expression of a different protein product.
  • a splicing factor such as Ul
  • DMD Duchenne muscular dystrophy
  • ESEs exonic splicing enhancers
  • RNA editing is a natural process whereby the diversity of gene products of a given sequence is increased by minor modification in the RNA.
  • the modification involves the conversion of adenosine (A) to inosine (I), resulting in an RNA sequence which is different from that encoded by the genome.
  • RNA modification is generally ensured by the ADAR enzyme, whereby the pre-RNA target forms an imperfect duplex RNA by base-pairing between the exon that contains the adenosine to be edited and an intronic non-coding element.
  • a classic example of A-I editing is the glutamate receptor GluR-B mRNA, whereby the change results in modified conductance properties of the channel (Higuchi M, et al. Cell. 1993;75: 1361-70).
  • transitions A ⁇ - >G or C ⁇ ->U changes
  • transversions any puring to any pyrimidine of vice versa
  • Transitions can be directly induced by using adening (ADARl/2), APOBEC) or cytosine deaminases (AID) which convert A to I or C to U, respectively.
  • Transversions can be indirectly induced by localizing reactive oxygen species damage to the bases of interest, which causes chemical modifications to be added to the affected bases, such as the conversion of guanine to oxo-guanine.
  • An oxo-gaunine is recognized as a T and will thus base pair with an adenine causing translation to be affected.
  • Proteins that can be recruited for ROS-mediated base damage include APEX and mini-SOG. With both approaches, these effectors can be fused to a catalytically inactive Casl3c and be recruited to sites on transcripts where these types of mutations are desired.
  • RNA targeting effector proteins of the present invention can be used to correct malfunctioning RNA modification.
  • RNA adenosine methylase (N(6)-methyladenosine) can be fused to the RNA targeting effector proteins of the invention and targeted to a transcript of interest.
  • This methylase causes reversible methylation, has regulatory roles and may affect gene expression and cell fate decisions by modulating multiple RNA-related cellular pathways (Fu et al Nat Rev Genet. 2014; 15(5):293-306).
  • Polyadenylation of an mRNA is important for nuclear transport, translation efficiency and stability of the mRNA, and all of these, as well as the process of polyadenylation, depend on specific RBPs. Most eukaryotic mRNAs receive a 3 ' poly(A) tail of about 200 nucleotides after transcription. Polyadenylation involves different RNA-binding protein complexes which stimulate the activity of a poly(A)polym erase (Minvielle-Sebastia L et al. Curr Opin Cell Biol. 1999; 1 1 :352- 7). It is envisaged that the RNA-targeting effector proteins provided herein can be used to interfere with or promote the interaction between the RNA-binding proteins and RNA.
  • OPMD oculopharyngeal muscular dystrophy
  • the mRNA is exported from the nucleus to the cytoplasm. This is ensured by a cellular mechanism which involves the generation of a carrier complex, which is then translocated through the nuclear pore and releases the mRNA in the cytoplasm, with subsequent recycling of the carrier.
  • mRNA localization ensures spatially regulated protein production. Localization of transcripts to a specific region of the cell can be ensured by localization elements.
  • the effector proteins described herein can be used to target localization elements to the RNA of interest.
  • the effector proteins can be designed to bind the target transcript and shuttle them to a location in the cell determined by its peptide signal tag. More particularly for instance, a RNA targeting effector protein fused to one or more nuclear localization signal (NLS) and/or one or more nuclear export signal (NES) can be used to alter RNA localization.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • localization signals include the zipcode binding protein (ZBP1) which ensures localization of ⁇ -actin to the cytoplasm in several asymmetric cell types, KDEL retention sequence (localization to endoplasmic reticulum), nuclear export signal (localization to cytoplasm), mitochondrial targeting signal (localization to mitochondria), peroxisomal targeting signal (localization to peroxisome) and m6A marking/YTHDF2 (localization to p-bodies).
  • ZBP1 zipcode binding protein
  • KDEL retention sequence localization to endoplasmic reticulum
  • nuclear export signal localization to cytoplasm
  • mitochondrial targeting signal localization to mitochondria
  • peroxisomal targeting signal localization to peroxisome
  • m6A marking/YTHDF2 localization to p-bodies.
  • Other approaches that are envisaged are fusion of the RNA targeting effector protein with proteins of known localization (for instance membrane, synapse).
  • the effector protein according to the invention may for instance be used in localization-dependent knockdown.
  • the effector protein By fusing the effector protein to a appropriate localization signal, the effector is targeted to a particular cellular compartment. Only target RNAs residing in this compartment will effectively be targeted, whereas otherwise identical targets, but residing in a different cellular compartment will not be targeted, such that a localization dependent knockdown can be established.
  • RNA targeting effector proteins described herein can be used to enhance or repress translation. It is envisaged that upregulating translation is a very robust way to control cellular circuits. Further, for functional studies a protein translation screen can be favorable over transcriptional upregulation screens, which have the shortcoming that upregulation of transcript does not translate into increased protein production.
  • RNA targeting effector proteins described herein can be used to bring translation initiation factors, such as EIF4G in the vicinity of the 5' untranslated repeat (5'UTR) of a messenger RNA of interest to drive translation (as described in De Gregorio et al. EMBO J. 1999; 18(17):4865-74 for a non-reprogrammable RNA binding protein).
  • translation initiation factors such as EIF4G
  • 5'UTR 5' untranslated repeat
  • GLD2 a cytoplasmic poly(A) polymerase
  • RNA targeting effector proteins envisaged herein can be used to block translational repressors of mRNA, such as ZBP1 (Huttelmaier S, et al. Nature. 2005;438:512-5). By binding to translation initiation site of a target RNA, translation can be directly affected.
  • RNA targeting effector proteins to a protein that stabilizes mRNAs, e.g. by preventing degradation thereof such as RNase inhibitors, it is possible to increase protein production from the transcripts of interest.
  • RNA targeting effector proteins described herein can be used to repress translation by binding in the 5UTR regions of a RNA transcript and preventing the ribosome from forming and beginning translation.
  • RNA targeting effector protein can be used to recruit Cafl, a component of the CCR4-NOT deadenylase complex, to the target mRNA, resulting in deadenylation or the target transcript and inhibition of protein translation.
  • the RNA targeting effector protein of the invention can be used to increase or decrease translation of therapeutically relevant proteins.
  • Examples of therapeutic applications wherein the RNA targeting effector protein can be used to downregulate or upregulate translation are in amyotrophic lateral sclerosis (ALS) and cardiovascular disorders.
  • ALS amyotrophic lateral sclerosis
  • Reduced levels of the glial glutamate transporter EAAT2 have been reported in ALS motor cortex and spinal cord, as well as multiple abnormal EAAT2 mRNA transcripts in ALS brain tissue. Loss of the EAAT2 protein and function thought to be the main cause of excitotoxicity in ALS. Restoration of EAAT2 protein levels and function may provide therapeutic benefit.
  • the RNA targeting effector protein can be beneficially used to upregulate the expression of EAAT2 protein, e.g. by blocking translational repressors or stabilizing mRNA as described above.
  • Apolipoprotein Al is the major protein component of high density lipoprotein (HDL) and ApoAl and HDL are generally considered as atheroprotective. It is envisages that the RNA targeting effector protein can be beneficially used to upregulate the expression of ApoAl, e.g. by blocking translational repressors or stabilizing mRNA as described above.
  • Translation is tightly coupled to mRNA turnover and regulated mRNA stability.
  • Specific proteins have been described to be involved in the stability of transcripts (such as the ELAV/Hu proteins in neurons, Keene JD, 1999, Proc Natl Acad Sci U S A. 96:5-7) and tristetraprolin (TTP). These proteins stabilize target mRNAs by protecting the messages from degradation in the cytoplasm (Peng SS et al., 1988, EMBO J. 17:3461-70).
  • RNA-targeting effector proteins of the present invention can be used to interfere with or to promote the activity of proteins acting to stabilize mRNA transcripts, such that mRNA turnover is affected.
  • recruitment of human TTP to the target RNA using the RNA targeting effector protein would allow for adenylate-uridylate-rich element (AU-rich element) mediated translational repression and target degradation.
  • AU-rich elements are found in the 3' UTR of many mRNAs that code for proto-oncogenes, nuclear transcription factors, and cytokines and promote RNA stability.
  • RNA targeting effector protein can be fused to HuR, another mRNA stabilization protein (Hinman MN and Lou H, Cell Mol Life Sci 2008;65:3168-81), and recruit it to a target transcript to prolong its lifetime or stabilize short-lived mRNA.
  • HuR HuR
  • another mRNA stabilization protein Hinman MN and Lou H, Cell Mol Life Sci 2008;65:3168-81
  • RNA-targeting effector proteins described herein can be used to promote degradation of target transcripts.
  • m6A methyltransferase can be recruited to the target transcript to localize the transcript to P-bodies leading to degradation of the target.
  • an RNA targeting effector protein as described herein can be fused to the non-specific endonuclease domain PilT N-terminus (PIN), to recruit it to a target transcript and allow degradation thereof.
  • PIN non-specific endonuclease domain
  • RNA-targeting effector proteins of the present invention can be used to interfere with the binding of auto-antibodies to mRNA transcripts.
  • DM1 dystrophy type 1
  • DMPK dystrophia myotonica-protein kinase
  • RNA-binding proteins bind to multiple sites on numerous RNAs to function in diverse processes.
  • the hnRNP Al protein has been found to bind exonic splicing silencer sequences, antagonizing the splicing factors, associate with telomere ends (thereby stimulating telomere activity) and bind miRNA to facilitate Drosha-mediated processing thereby affecting maturation.
  • the RNA-binding effector proteins of the present invention can interfere with the binding of RNA-binding proteins at one or more locations.
  • RNA adopts a defined structure in order to perform its biological activities. Transitions in conformation among alternative tertiary structures are critical to most RNA-mediated processes. However, RNA folding can be associated with several problems. For instance, RNA may have a tendency to fold into, and be upheld in, improper alternative conformations and/or the correct tertiary structure may not be sufficiently thermodynamically favored over alternative structures.
  • the RNA targeting effector protein, in particular a cleavage-deficient or dead RNA targeting protein, of the invention may be used to direct folding of (m)RNA and/or capture the correct tertiary structure thereof.
  • Casl3c in a complex with crRNA is activated upon binding to target RNA and subsequently cleaves any nearby ssRNA targets (i.e. "collateral” or “bystander” effects).
  • Casl3c once primed by the cognate target, can cleave other (non-complementary) RNA molecules. Such promiscuous RNA cleavage could potentially cause cellular toxicity, or otherwise affect cellular physiology or cell status.
  • the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of cell dormancy. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of cell cycle arrest. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in reduction of cell growth and/or cell proliferation. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of cell anergy.
  • the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of cell apoptosis. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in incuction of cell necrosis. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of cell death. In certain embodiments, the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein are used for or are for use in induction of programmed cell death.
  • the invention relates to a method for induction of cell dormancy comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for induction of cell cycle arrest comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for reduction of cell growth and/or cell proliferation comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for induction of cell anergy comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for induction of cell apoptosis comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for induction of cell necrosis comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates to a method for induction of cell death comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein. In certain embodiments, the invention relates to a method for induction of programmed cell death comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the methods and uses as described herein may be therapeutic or prophylactic and may target particular cells, cell (sub)populations, or cell/tissue types.
  • the methods and uses as described herein may be therapeutic or prophylactic and may target particular cells, cell (sub)populations, or cell/tissue types expressing one or more target sequences, such as one or more particular target RNA (e.g. ss RNA).
  • target cells may for instance be cancer cells expressing a particular transcript, e.g. neurons of a given class, (immune) cells causing e.g. autoimmunity, or cells infected by a specific (e.g. viral) pathogen, etc.
  • the invention relates to a method for treating a pathological condition characterized by the presence of undersirable cells (host cells), comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the invention relates the use of the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for treating a pathological condition characterized by the presence of undersirable cells (host cells).
  • the invention relates the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for use in treating a pathological condition characterized by the presence of undersirable cells (host cells).
  • the invention relates to the use of the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for treating, preventing, or alleviating cancer.
  • the invention relates to the non- naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for use in treating, preventing, or alleviating cancer.
  • the invention relates to a method for treating, preventing, or alleviating cancer comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein.
  • the CRISPR-Cas system targets a target specific for the cancer cells.
  • the invention relates to the use of the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for treating, preventing, or alleviating infection of cells by a pathogen.
  • the invention relates to the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for use in treating, preventing, or alleviating infection of cells by a pathogen.
  • the invention relates to a method for treating, preventing, or alleviating infection of cells by a pathogen comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein. It is to be understood that preferably the CRISPR-Cas system targets a target specific for the cells infected by the pathogen (e.g. a pathogen derived target). In certain embodiments, the invention relates to the use of the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for treating, preventing, or alleviating an autoimmune disorder.
  • the invention relates to the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein for use in treating, preventing, or alleviating an autoimmune disorder.
  • the invention relates to a method for treating, preventing, or alleviating an autoimmune disorder comprising introducing or inducing the non-naturally occurring or engineered composition, vector system, or delivery systems as derscribed herein. It is to be understood that preferably the CRISPR-Cas system targets a target specific for the cells responsible for the autoimmune disorder (e.g. specific immune cells).
  • RNA targeting effector protein can be used for detection of nucleic acids or proteins in a biological sample.
  • the samples can be can be cellular or cell-free.
  • Northern blotting involves the use of electrophoresis to separate RNA samples by size.
  • the RNA targeting effector protein can be used to specifically bind and detect the target RNA sequence.
  • a RNA targeting effector protein can also be fused to a fluorescent protein (such as GFP) and used to track RNA localization in living cells. More particularly, the RNA targeting effector protein can be inactivated in that it no longer cleaves RNA.
  • a split RNA targeting effector protein can be used, whereby the signal is dependent on the binding of both subproteins, in order to ensure a more precise visualization.
  • a split fluorescent protein can be used that is reconstituted when multiple RNA targeting effector protein complexes bind to the target transcript. It is further envisaged that a transcript is targeted at multiple binding sites along the mRNA so the fluorescent signal can amplify the true signal and allow for focal identification.
  • the fluorescent protein can be reconstituted form a split intein.
  • RNA targeting effector proteins are for instance suitably used to determine the localization of the RNA or specific splice variants, the level of mRNA transcript, up- or down regulation of transcripts and disease-specific diagnosis.
  • the RNA targeting effector proteins can be used for visualization of RNA in (living) cells using e.g. fluorescent microscopy or flow cytometry, such as fluorescence-activated cell sorting (FACS) which allows for high-throughput screening of cells and recovery of living cells following cell sorting. Further, expression levels of different transcripts can be assessed simultaneously under stress, e.g. inhibition of cancer growth using molecular inhibitors or hypoxic conditions on cells. Another application would be to track localization of transcripts to synaptic connections during a neural stimulus using two photon microscopy.
  • the components or complexes according to the invention as described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015; 348(6233)), such as for instance with (fluorescently) labeled Casl3c effectors.
  • MEFISH error-robust fluorescence in situ hybridization
  • RNA targeting effector protein of the invention can for instance be used to target a probe to a selected RNA sequence.
  • the invention provides agents and methods for diagnosing and monitoring health states through non-invasive sampling of cell free RNA, including testing for risk and guiding RNA- targeted therapies, and is useful in setting where rapid administration of therapy is important to treatment outcomes.
  • the invention provides cancer detection methods and agents for circulating tumor RNA, including for monitoring recurrence and/or development of common drug resistance mutations.
  • the invention provides detection methods and agents for detection and/or identification of bacterial species directly from blood or serum to monitor, e.g., disease progression and sepsis.
  • the Casl3c proteins and derivative are used to distinguish and diagnose common diseases such as rhinovirus or upper respiratory tract infections from more serious infections such as bronchitis.
  • the invention provides methods and agents for rapid genotyping for emergency pharmacogenomics, including guidance for administration of anticoagulants during myocardial infarction or stroke treatment based on, e.g., VKORC1, CYP2C9, and CYP2C19 genotyping.
  • the invention provides agents and methods for monitoring food contamination by bacteria at all points along a food production and delivery chain.
  • the invention provides for quality control and monitoring, e.g. by identification of food sources and determination of purity.
  • the invention may be used to identify or confirm a food sources, such as a species of animal meat and seafood.
  • the invention is used in phorensic determinations.
  • phorensic determinations For example, crime scene samples containing blood or other bodily fluids.
  • the invention is used to identify nucleic acid samples from fingerprints.
  • RNA-targeting effector protein in RNA origami/in vitro assembly lines - combinatorics
  • RNA origami refers to nanoscale folded structures for creating two-dimensional or three-dimensional structures using RNA as integrated template.
  • the folded structure is encoded in the RNA and the shape of the resulting RNA is thus determined by the synthesized RNA sequence (Geary, et al. 2014. Science, 345 (6198). pp. 799-804).
  • the RNA origami may act as scaffold for arranging other components, such as proteins, into complexes.
  • the RNA targeting effector protein of the invention can for instance be used to target proteins of interest to the RNA origami using a suitable guide RNA.
  • RNA-targeting effector protein in RNA isolation or purification, enrichment or depletion
  • RNA targeting effector protein when complexed to RNA can be used to isolate and/or purify the RNA.
  • the RNA targeting effector protein can for instance be fused to an affinity tag that can be used to isolate and/or purify the RNA-RNA targeting effector protein complex.
  • affinity tag can be used to isolate and/or purify the RNA-RNA targeting effector protein complex.
  • Such applications are for instance useful in the analysis of gene expression profiles in cells.
  • the RNA targeting effector proteins can be used to target a specific noncoding RNA (ncRNA) thereby blocking its activity, providing a useful functional probe.
  • the effecetor protein as described herein may be used to specifically enrich for a particular RNA (including but not limited to increasing stability, etc.), or alternatively to specifically deplete a particular RNA (such as without limitation for instance particular splice variants, isoforms, etc.).
  • RNA knockdown strategies such as siRNA have the disadvantage that they are mostly limited to targeting cytosolic transcripts since the protein machinery is cytosolic.
  • the advantage of a RNA targeting effector protein of the present invention an exogenous system that is not essential to cell function, is that it can be used in any compartment in the cell. By fusing a NLS signal to the RNA targeting effector protein, it can be guided to the nucleus, allowing nuclear RNAs to be targeted. It is for instance envisaged to probe the function of lincRNAs. Long intergenic non-coding RNAs (lincRNAs) are a vastly underexplored area of research. Most lincRNAs have as of yet unknown functions which could be studies using the RNA targeting effector protein of the invention.
  • RNA targeting effector protein of the invention can be designed to recruit a biotin ligase to a specific transcript in order to label locally bound proteins with biotin. The proteins can then be pulled down and analyzed by mass spectrometry to identify them.
  • RNA targeting effector proteins of the invention can further be used to assemble complexes on RNA. This can be achieved by functionalizing the RNA targeting effector protein with multiple related proteins (e.g. components of a particular synthesis pathway). Alternatively, multiple RNA targeting effector proteins can be functionalized with such different related proteins and targeted to the same or adjacent target RNA. Useful application of assembling complexes on RNA are for instance facilitating substrate shuttling between proteins.
  • RNA targeting effector proteins of the invention can be used fused to split proteins of toxic domains for targeted cell death, for instance using cancer-linked RNA as target transcript.
  • pathways involving protein-protein interaction can be influenced in synthetic biological systems with e.g. fusion complexes with the appropriate effectors such as kinases or other enzymes.
  • Protein splicing is a post-translational process in which an intervening polypeptide, referred to as an intein, catalyzes its own excision from the polypeptides flacking it, referred to as exteins, as well as subsequent ligation of the exteins.
  • the assembly of two or more RNA targeting effector proteins as described herein on a target transcript could be used to direct the release of a split intein (Topilina and Mills Mob DNA. 2014 Feb 4;5(1):5), thereby allowing for direct computation of the existence of a mRNA transcript and subsequent release of a protein product, such as a metabolic enzyme or a transcription factor (for downstream actuation of transcription pathways).
  • This application may have significant relevance in synthetic biology (see above) or large-scale bioproduction (only produce product under certain conditions).
  • fusion complexes comprising an RNA targeting effector protein of the invention and an effector component are designed to be inducible, for instance light inducible or chemically inducible. Such inducibility allows for activation of the effector component at a desired moment in time.
  • Light inducibility is for instance achieved by designing a fusion complex wherein CRY2PHR/CIBN pairing is used for fusion. This system is particularly useful for light induction of protein interactions in living cells (Konermann S, et al. Nature. 2013;500:472-476).
  • the RNA targeting effector protein of the inventions can be modulated by inducible promoters, such as tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system), hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system and an arabinose-inducible gene expression system.
  • inducible promoters such as tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system)
  • hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system and an arabinose-inducible gene expression system.
  • expression of the RNA targeting effector protein can be modulated via a riboswitch, which can sense a small molecule like tetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).
  • the delivery of the RNA targeting effector protein of the invention can be modulated to change the amount of protein or crRNA in the cell, thereby changing the magnitude of the desired effect or any undesired off-target effects.
  • the RNA targeting effector proteins described herein can be designed to be self-inactivating.
  • RNA either mRNA or as a replication RNA therapeutic (Wrobleska et al Nat Biotechnol. 2015 Aug; 33(8): 839-841)
  • they can self- inactivate expression and subsequent effects by destroying the own RNA, thereby reducing residency and potential undesirable effects.
  • RNA targeting effector proteins as described herein, reference is made to Mackay JP et al (Nat Struct Mol Biol. 2011 Mar; 18(3):256-61), Nelles et al (Bioessays. 2015 Jul; 37(7): 732-9) and Abil Z and Zhao H (Mol Biosyst. 2015 Oct; l 1(10):2658- 65), which are incorporated herein by reference.
  • the following applications are envisaged in certain embodiments of the invention, preferably in certain embodiments by using catalytically inactive Casl3c: enhancing translation (e.g. Casl3c - translation promotion factor fusions (e.g.
  • eIF4 fusions repressing translation
  • repressing translation e.g. gRNA targeting ribosome binding sites
  • exon skipping e.g. gRNAs targeting splice donor and/or acceptor sites
  • exon inclusion e.g. gRNA targeting a particular exon splice donor and/or acceptor site to be included or Casl3c fused to or recruiting spliceosome components (e.g. Ul snRNA)
  • accessing RNA localization e.g. Casl3c - marker fusions (e.g.EGFP fusions)
  • altering RNA localization e.g. Casl3c - localization signal fusions (e.g.
  • RNA degradation in this case no catalytically inactive Casl3c is to be used if relied on the activity of Casl3c, alternatively and for increased specificity, a split Casl3c may be used; inhibition of non-coding RNA function (e.g. miRNA), such as by degradation or binding of gRNA to functional sites (possibly titrating out at specific sites by relocalization by Casl3c-signal sequence fusions).
  • non-coding RNA function e.g. miRNA
  • Casl3c is capable of mediating resistance to RNA phages. It is therefore envisaged that Casl3c can be used to immunize, e.g. 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.
  • retroviruses e.g lentiviruses, such as HIV
  • HCV lentiviruses, such as HIV
  • Ebola virus Zika virus.
  • Casl3c can process (cleave) its own array. This applies to both the wildtype Casl3c protein and mutated Casl3c protein containing one or more mutated amino acid residues R597, H602, R1278 and H1283, relative to D2c2 residues, such as one or more of the modifications selected from R597A, H602A, R1278A and H1283A. It is therefore envisaged that multiple crRNAs designed for different target transcripts and/or applications can be delivered as a single pre-crRNA or as a single transcript driven by one promotor. Such method of delivery has the advantages that it is substantially more compact, easier to synthesize and easier to delivery in viral systems.
  • amino acid numbering as described herein refers to Lsh C2c2 protein. It will be understood that exact amino acid positions may vary for orthologues of Lsh C2c2, which can be adequately determined by protein alignment, as is known in the art, and as described herein elsewhere.
  • compositions and systems described herein in genome or transcriptome engineering, e.g. for altering or manipulating the (protein) expression of one or more genes or the one or more gene products, in prokaryotic or eukaryotic cells, in vitro, in vivo or ex vivo.
  • the invention provides methods and compositions for modulating, e.g., reducing, (protein) expression of a target RNA in cells.
  • a Casl3c system of the invention is provided that interferes with transcription, stability, and / or translation of an RNA.
  • an effective amount of Casl3c system is used to cleave RNA or otherwise inhibit RNA expression.
  • the system has uses similar to siRNA and shRNA, thus can also be substituted for such methods.
  • the method includes, without limitation, use of a Casl3c system as a substitute for e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA.
  • the Casl3c system is introduced into a target cell, e.g., by being administered to a mammal that includes the target cell,
  • a Casl3c system of the invention is specific.
  • interfering ribonucleic acid such as an siRNA or shRNA
  • a Casl3c system of the invention can be designed with high specificity.
  • the effecteor protein (CRISPR enzyme; Casl3c) according to the invention as described herein is associated with or fused to a destabilization domain (DD).
  • the DD is ER50.
  • a corresponding stabilizing ligand for this DD is, in some embodiments, 4HT.
  • one of the at least one DDs is ER50 and a stabilizing ligand therefor is 4HT.
  • CMP8 In some embodiments, the DD is DHFR50.
  • a corresponding stabilizing ligand for this DD is, in some embodiments, TMP.
  • one of the at least one DDs is DHFR50 and a stabilizing ligand therefor is TMP.
  • the DD is ER50.
  • a corresponding stabilizing ligand for this DD is, in some embodiments, CMP8.
  • CMP8 may therefore be an alternative stabilizing ligand to 4HT in the ER50 system. While it may be possible that CMP8 and 4HT can/should be used in a competitive matter, some cell types may be more susceptible to one or the other of these two ligands, and from this disclosure and the knowledge in the art the skilled person can use CMP8 and/or 4HT.
  • one or two DDs may be fused to the N- terminal end of the CRISPR enzyme with one or two DDs fused to the C- terminal of the CRISPR enzyme.
  • the at least two DDs are associated with the CRISPR enzyme and the DDs are the same DD, i.e. the DDs are homologous.
  • both (or two or more) of the DDs could be ER50 DDs. This is preferred in some embodiments.
  • both (or two or more) of the DDs could be DHFR50 DDs. This is also preferred in some embodiments.
  • the at least two DDs are associated with the CRISPR enzyme and the DDs are different DDs, i.e. the DDs are heterologous.
  • one of the DDS could be ER50 while one or more of the DDs or any other DDs could be DHFR50. Having two or more DDs which are heterologous may be advantageous as it would provide a greater level of degradation control.
  • a tandem fusion of more than one DD at the N or C-term may enhance degradation; and such a tandem fusion can be, for example ER50-ER50-Casl3c or DHFR-DHFR-Cas 13c It is envisaged that high levels of degradation would occur in the absence of either stabilizing ligand, intermediate levels of degradation would occur in the absence of one stabilizing ligand and the presence of the other (or another) stabilizing ligand, while low levels of degradation would occur in the presence of both (or two of more) of the stabilizing ligands. Control may also be imparted by having an N-terminal ER50 DD and a C-terminal DHFR50 DD.
  • the fusion of the CRISPR enzyme with the DD comprises a linker between the DD and the CRISPR enzyme.
  • the linker is a GlySer linker.
  • the DD-CRISPR enzyme further comprises at least one Nuclear Export Signal (NES).
  • the DD-CRISPR enzyme comprises two or more NESs.
  • the DD-CRISPR enzyme comprises at least one Nuclear Localization Signal (NLS). This may be in addition to an NES.
  • the CRISPR enzyme comprises or consists essentially of or consists of a localization (nuclear import or export) signal as, or as part of, the linker between the CRISPR enzyme and the DD.
  • HA or Flag tags are also within the ambit of the invention as linkers. Applicants use NLS and/or NES as linker and also use Glycine Serine linkers as short as GS up to (GGGGS) (SEQ ID No. 20)
  • Destabilizing domains have general utility to confer instability to a wide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar 7, 2012; 134(9): 3942-3945, incorporated herein by reference.
  • CMP8 or 4-hydroxytamoxifen can be destabilizing domains. More generally, A temperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizing residue by the N-end rule, was found to be stable at a permissive temperature but unstable at 37 °C. The addition of methotrexate, a high-affinity ligand for mammalian DHFR, to cells expressing DHFRts inhibited degradation of the protein partially.
  • methotrexate a high-affinity ligand for mammalian DHFR
  • a rapamycin derivative was used to stabilize an unstable mutant of the FRB domain of mTOR (FRB*) and restore the function of the fused kinase, GSK-3p.6,7
  • FRB* FRB domain of mTOR
  • GSK-3p.6,7 This system demonstrated that ligand-dependent stability represented an attractive strategy to regulate the function of a specific protein in a complex biological environment.
  • a system to control protein activity can involve the DD becoming functional when the ubiquitin complementation occurs by rapamycin induced dimerization of FK506-binding protein and FKBP12.
  • Mutants of human FKBP12 or ecDHFR protein can be engineered to be metabolically unstable in the absence of their high-affinity ligands, Shield-1 or trimethoprim (TMP), respectively. These mutants are some of the possible destabilizing domains (DDs) useful in the practice of the invention and instability of a DD as a fusion with a CRISPR enzyme confers to the CRISPR protein degradation of the entire fusion protein by the proteasome. Shield- 1 and TMP bind to and stabilize the DD in a dose-dependent manner.
  • the estrogen receptor ligand binding domain (ERLBD, residues 305-549 of ERS1) can also be engineered as a destabilizing domain.
  • the mutant ERLBD can be fused to a CRISPR enzyme and its stability can be regulated or perturbed using a ligand, whereby the CRISPR enzyme has a DD.
  • Another DD can be a 12-kDa (107-amino-acid) tag based on a mutated FKBP protein, stabilized by Shieldl ligand; see, e.g., Nature Methods 5, (2008).
  • a DD can be a modified FK506 binding protein 12 (FKBP12) that binds to and is reversibly stabilized by a synthetic, biologically inert small molecule, Shield-1; see, e.g., Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell. 2006;126:995-1004; Banaszynski LA, Sellmyer MA, Contag CH, Wandless TJ, Thorne SH. Chemical control of protein stability and function in living mice. Nat Med.
  • FKBP12 modified FK506 binding protein 12
  • the knowledge in the art includes a number of DDs, and the DD can be associated with, e.g., fused to, advantageously with a linker, to a CRISPR enzyme, whereby the DD can be stabilized in the presence of a ligand and when there is the absence thereof the DD can become destabilized, whereby the CRISPR enzyme is entirely destabilized, or the DD can be stabilized in the absence of a ligand and when the ligand is present the DD can become destabilized; the DD allows the CRISPR enzyme and hence the CRISPR-Cas complex or system to be regulated or controlled— turned on or off so to speak, to thereby provide means for regulation or control of the system, e.g., in an in vivo or in vitro environment.
  • a protein of interest when expressed as a fusion with the DD tag, it is destabilized and rapidly degraded in the cell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads to a D associated Cas being degraded.
  • a new DD When fused to a protein of interest, its instability is conferred to the protein of interest, resulting in the rapid degradation of the entire fusion protein. Peak activity for Cas is sometimes beneficial to reduce off-target effects. Thus, short bursts of high activity are preferred.
  • the present invention is able to provide such peaks. In some senses the system is inducible. In some other senses, the system repressed in the absence of stabilizing ligand and de-repressed in the presence of stabilizing ligand.
  • the term "plant” relates to any various photosynthetic, eukaryotic, unicellular or multicellular organism of the kingdom Plantae characteristically growing by cell division, containing chloroplasts, and having cell walls comprised of cellulose.
  • the term plant encompasses monocotyledonous and dicotyledonous plants.
  • the plants are intended to comprise without limitation angiosperm and gymnosperm plants such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, Brussel' s sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, Clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, mai
  • RNA targetingRNA targeting system as described herein can be used to confer desired traits on essentially any plant.
  • a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above.
  • target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis).
  • crops including grain crops e.g., wheat, maize, rice, millet, barley
  • the methods and CRISPR-Cas systems can be used over a broad range of plants, such as for example with dicotyledonous plants belonging to the orders Magniolales, Illiciales, Laurales, Piperaies, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvaies, Urticales, Lecythidaies, Violales, Saiicaies, Capparaies, Ericaies, Diapensales, Ebenales, Primulales, Resales, Fabales, Podostemales, Haloragales, Myrtales, Comales, Proteales, San tales, Rafflesiaies, Celastrales, Euphorbiaies, Rhamnaies, Sapin
  • R A targetingRNA targeting CRISPR systems and methods of use described herein can be used over a broad range of plant species, included in the non-limitative list of dicot, monocot or gyrnnosperm genera hereunder: Atropa, Alseodaphne, Anacardhim, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Cqffea, C curbit , Daucus, Duguetia, Eschscholzia, Incus, Fragaria, Glauchim, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea
  • RNA targeting CRISPR systems and methods of use can also be used over a broad range of "algae” or “algae cells”; including for example algea selected from several eukaryotic phyla, including the Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as well as the prokaryotic phylum Cyanobacteria (blue-green algae).
  • algea selected from several eukaryotic phyla including the Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as well as the prokaryotic phylum Cyanobacteria (blue-green algae).
  • algae includes for example algae selected from : Amphora, Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris, Nannnochloropsis, Navicula, Nephrochions, Nephroselmis, Nitzschia, Nodulana, Nostoc, Oochromonas, Oocystis, Osciilartoria, Pavlova, Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetrasel
  • Plant tissue A part of a plant, i.e., a "plant tissue” may be treated according to the methods of the present invention to produce an improved plant.
  • Plant tissue also encompasses plant cells.
  • plant cell refers to individual units of a living plant, either in an intact whole plant or in an isolated form grown in in vitro tissue cultures, on media or agar, in suspension in a growth media or buffer or as a part of higher organized unites, such as, for example, plant tissue, a plant organ, or a whole plant.
  • a "protoplast” refers to a plant cell that has had its protective cell wall completely or partially removed using, for example, mechanical or enzymatic means resulting in an intact biochemical competent unit of living plant that can reform their cell wall, proliferate and regenerate grow into a whole plant under proper growing conditions.
  • transformation broadly refers to the process by which a plant host is genetically modified by the introduction of DNA by means of Agrobacteria or one of a variety of chemical or physical methods.
  • plant host refers to plants, including any cells, tissues, organs, or progeny of the plants.
  • plant tissues or plant cells can be transformed and include, but are not limited to, protoplasts, somatic embryos, pollen, leaves, seedlings, stems, calli, stolons, microtubers, and shoots.
  • a plant tissue also refers to any clone of such a plant, seed, progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed.
  • the term "transformed” as used herein refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced.
  • the introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is transmitted to the subsequent progeny.
  • the "transformed” or “transgenic” cell or plant may also include progeny of the cell or plant and progeny produced from a breeding program employing such a transformed plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of the introduced DNA molecule.
  • the transgenic plant is fertile and capable of transmitting the introduced DNA to progeny through sexual reproduction.
  • progeny such as the progeny of a transgenic plant
  • the introduced DNA molecule may also be transiently introduced into the recipient cell such that the introduced DNA molecule is not inherited by subsequent progeny and thus not considered “transgenic”.
  • a non-transgenic plant or plant cell is a plant which does not contain a foreign DNA stably integrated into its genome.
  • plant promoter is a promoter capable of initiating transcription in plant cells, whether or not its origin is a plant cell.
  • exemplary suitable plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobium which comprise genes expressed in plant cells.
  • a "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 may include yeasts, molds, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell.
  • yeast cell refers to any fungal cell within the phyla Ascomycota and Basidiomycota.
  • Yeast cells may include budding yeast cells, fission yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in laboratory and industrial settings are part of the phylum Ascomycota.
  • the yeast cell is an S. cerervisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell.
  • Other yeast cells may include without limitation Candida spp. (e.g., Candida albicans), Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp.
  • the fungal cell is a filamentous fungal cell.
  • filamentous fungal cell refers to any type of fungal cell that grows in filaments, i.e., hyphae or mycelia.
  • filamentous fungal cells may 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).
  • the fungal cell is an industrial strain.
  • industrial strain refers to any strain of fungal cell used in or isolated from an industrial process, e.g., production of a product on a commercial or industrial scale.
  • Industrial strain may refer to a fungal species that is typically used in an industrial process, or it may refer to an isolate of a fungal species that may be also used for non-industrial purposes (e.g., laboratory research).
  • industrial processes may include fermentation (e.g., in production of food or beverage products), distillation, biofuel production, production of a compound, and production of a polypeptide.
  • industrial strains may include, without limitation, JAY270 and ATCC4124.
  • the fungal cell is a polyploid cell.
  • a "polyploid" cell may refer to any cell whose genome is present in more than one copy.
  • a polyploid cell may refer to a type of cell that is naturally found in a polyploid state, or it may refer to a cell that has been induced to exist in a polyploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication).
  • a polyploid cell may refer to a cell whose entire genome is polyploid, or it may refer to a cell that is polyploid in a particular genomic locus of interest.
  • guideRNA may more often be a rate-limiting component in genome engineering of polyploid cells than in haploid cells, and thus the methods using the Casl3c CRISPRS system described herein may take advantage of using a certain fungal cell type.
  • the fungal cell is a diploid cell.
  • a diploid cell may refer to any cell whose genome is present in two copies.
  • a diploid cell may refer to a type of cell that is naturally found in a diploid state, or it may refer to a cell that has been induced to exist in a diploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication).
  • the S. cerevisiae strain S228C may be maintained in a haploid or diploid state.
  • a diploid cell may refer to a cell whose entire genome is diploid, or it may refer to a cell that is diploid in a particular genomic locus of interest.
  • the fungal cell is a haploid cell.
  • a "haploid" cell may refer to any cell whose genome is present in one copy.
  • a haploid cell may refer to a type of cell that is naturally found in a haploid state, or it may refer to a cell that has been induced to exist in a haploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S.
  • a haploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell that is haploid in a particular genomic locus of interest.
  • yeast expression vector refers to a nucleic acid that contains one or more sequences encoding an RNA and/or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell.
  • yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in 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 may contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers).
  • CEN centromeric
  • ARS autonomous replication sequence
  • a promoter such as an RNA Polymerase III promoter
  • a terminator such as an RNA polymerase III terminator
  • an origin of replication e.g., auxotrophic, antibiotic, or other selectable markers
  • marker gene e.g., auxotrophic, antibiotic, or other selectable markers.
  • expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2 ⁇ plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and
  • RNA targeting CRISP system components in the genome of plants and plant cells
  • the polynucleotides encoding the components of the RNA targeting CRISPR system are introduced for stable integration into the genome of a plant cell.
  • the design of the transformation vector or the expression system can be adjusted depending on when, where and under what conditions the guide RNA and/or the RNA targeting gene(s) are expressed.
  • RNA targeting CRISPR system stably into the genomic DNA of a plant cell.
  • RNA targeting CRISPR system for stable integration into the DNA of a plant organelle such as, but not limited to a plastid, e mitochondrion or a chloroplast.
  • the 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 the guide RNA and/or RNA targeting enzyme in a plant cell; a 5' untranslated region to enhance expression ; an intron element to further enhance expression in certain cells, such as monocot cells; a multiple- cloning site to provide convenient restriction sites for inserting the one or more guide RNAs and/or the RNA targeting gene sequences and other desired elements; and a 3' untranslated region to provide for efficient termination of the expressed transcript.
  • a promoter element that can be used to express the guide RNA and/or RNA targeting enzyme in a plant cell
  • a 5' untranslated region to enhance expression an intron element to further enhance expression in certain cells, such as monocot cells
  • a multiple- cloning site to provide convenient restriction sites for inserting the one or more guide RNAs and/or the RNA targeting gene sequences and other desired elements
  • a 3' untranslated region to provide
  • the elements of the expression system may be on one or more expression constructs which are either circular such as a plasmid or transformation vector, or non-circular such as linear double stranded DNA.
  • RNA targeting CRISPR expression system comprises at least:
  • gRNA guide RNA
  • components (a) or (b) are located on the same or on different constructs, and whereby the different nucleotide sequences can be under control of the same or a different regulatory element operable in a plant cell.
  • DNA construct(s) containing the components of the RNA targeting CRISPR system, and, where applicable, template sequence may be introduced into the genome of a plant, plant part, or plant cell by a variety of conventional techniques.
  • the process generally comprises the steps of selecting a suitable host cell or host tissue, introducing the construct(s) into the host cell or host tissue, and regenerating plant ceils or plants therefrom.
  • the DNA construct may be introduced into the plant cell using techniques such as but not limited to eiectroporation, microinjection, aerosol beam injection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see also Fu et al ,, Transgenic Res, 2000 Feb;9(l): l l-9).
  • DNA particle bombardment see also Fu et al ,, Transgenic Res, 2000 Feb;9(l): l l-9.
  • the basis of particle bombardment is the acceleration of particles coated with gene/s of interest toward cells, resulting in the penetration of the protoplasm by the particles and typically stable integration into the genome, (see e.g. Klein et al, Nature ( 1987), Klein et al, Bio/Technology (1992), Casas et al, Proc. Natl. Acad, Sci. USA (1993),).
  • the DNA constructs containing components of the RNA targeting CRISPR system may be introduced into the plant by Agrobacterium -mediated transformation.
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium iumefaciens host vector.
  • the foreign DNA can be incorporated into the genome of plants by infecting the plants or by incubating plant protoplasts with Agrobacterium bacteria, containing one or more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055).
  • CRISPR system described herein are typically placed under control of a plant promoter, i.e. a promoter operable in plant cells.
  • a plant promoter i.e. a promoter operable in plant cells.
  • the use of different types of promoters is envisaged.
  • a constitutive plant promoter is a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as "constitutive expression").
  • ORF open reading frame
  • One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter.
  • the present invention envisages methods for modifying RNA sequences and as such also envisages regulating expression of plant biomolecules. In particular embodiments of the present invention it is thus advantageous to place one or more elements of the RNA targeting CRISPR system under the control of a promoter that can be regulated.
  • RNA targeting CRISPR components are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter issue-preferred promoters can be utilized to target enhanced expression in certain cell types within a particular plant tissue, for instance vascular cells in leaves or roots or in specific cells of the seed.
  • promoters that are inducible and that allow for spatiotemporal control of gene editing or gene expression may use a form of energy.
  • the form of energy may include but is not limited to sound energy, electromagnetic radiation, chemical energy and/or thermal energy.
  • inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome)., such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner.
  • LITE Light Inducible Transcriptional Effector
  • the components of a light inducible system may include a RNA targeting CRISPR enzyme, a light- responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain.
  • a RNA targeting CRISPR enzyme e.g. from Arabidopsis thaliana
  • a light- responsive cytochrome heterodimer e.g. from Arabidopsis thaliana
  • transcriptional activation/repression domain e.g. from Arabidopsis thaliana
  • transient or inducible expression can be achieved by using, for example, chemical -regulated promotors, i .e. whereby the application of an exogenous chemical induces gene expression. Modulating of gene expression can also be obtained by a chemical- repressible promoter, where application of the chemical represses gene expression.
  • Chemical- inducible promoters include, but are not limited to, the maize ln2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST 1-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1 a promoter (Ono et al,, (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid.
  • Promoters which are regulated by antibiotics such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991 ) Moi Gen Genet 227:229-37; U.S. Patent Nos. 5,814,618 and 5,789, 156) can also be used herein.
  • Translocation to and/or expression in specific plant organelles such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991 ) Moi Gen Genet 227:229-37; U.S. Patent Nos. 5,814,618 and 5,789, 156) can also be used herein.
  • the expression system may comprise elements for translocation to and/or expression in a specific plant organelle.
  • the RNA targeting CRISPR system is used to specifically modify expression and/or translation of chloroplast genes or to ensure expression in the chloroplast.
  • use is made of chloroplast transformation methods or proximityalization of the RNA targeting CRISPR components to the chloroplast.
  • the introduction of genetic modifications in the plastid genome can reduce biosafety issues such as gene flow through pollen.
  • Methods of chloroplast transformation are known in the art and include Particle bombardment, PEG treatment, and microinjection. Additionally, methods involving the translocation of transformation cassettes from the nuclear genome to the plastid can be used as described in WO2010061 186.
  • RNA targeting CRISPR components it is envisaged to target one or more of the RNA targeting CRISPR components to the plant chloroplast.
  • This is achieved by incorporating in the expression construct a sequence encoding a chloroplast transit peptide (CTP) or plastid transit peptide, operably linked to the 5' region of the sequence encoding the RNA targeting protein.
  • CTP chloroplast transit peptide
  • the CTP is removed in a processing step during translocation into the chloroplast.
  • Chloroplast targeting of expressed proteins is well known to the skilled artisan (see for instance Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61 : 157-180) .
  • Transgenic algae may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol) or other products. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.
  • RNA targeting protein and guide RNA(s) are introduced in algae expressed using a vector that expresses RNA targeting protein under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2 -tubulin.
  • Guide RNA is optionally delivered using a vector containing T7 promoter.
  • RNA targeting mRNA and in vitro transcribed guide RNA can be delivered to algal cells. Electroporation protocols are available to the skilled person such as the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.
  • the invention relates to the use of the RNA targeting CRISPR system for RNA editing in yeast cells.
  • Methods for transforming yeast cells which can be used to introduce polynucleotides encoding the RNA targeting CRISPR system components are well known to the artisan and are 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 by electroporation.
  • the guide RNA and/or RNA targeting gene are transiently expressed in the plant cell.
  • the RNA targeting CRISPR system can ensure modification of RNA target molecules only when both the guide RNA and the RNA. targeting protein is present in a cell, such that gene expression can further be controlled.
  • the expression of the RNA targeting enzyme is transient, plants regenerated from such plant cells typically contain no foreign DNA.
  • the RNA targeting enzyme is stably expressed by the plant cell and the guide sequence is transiently expressed.
  • the RNA targeting CRISPR system components can be introduced in the plant cells using a plant viral vector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996;34:299-323).
  • said viral vector is a vector from a DNA virus.
  • gemini virus 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
  • nanovirus e.g., Faba bean necrotic yellow virus.
  • said viral vector is a vector from an RNA virus.
  • tobravirus e.g., tobacco rattle virus, tobacco mosaic virus
  • potexvirus e.g., potato virus X
  • hordeivirus e.g., barley stripe mosaic virus
  • the vector used for transient expression of RNA targeting CRISPR constructs is for instance a pEAQ vector, which is tailored for Agrobacterium-mediated transient expression (Sainsbury F. et al., Plant Biotechnol J. 2009 Sep;7(7):682-93) in the protoplast. Precise targeting of genomic locations was demonstrated using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing a CRISPR enzyme (Scientific Reports 5, Article number; 14926 (2015), doi : 10.1038/srepl 4926).
  • CaLCuV Cabbage Leaf Curl virus
  • double-stranded DNA fragments encoding the guide RNA and/or the RNA targeting gene can be transiently introduced into the plant cell.
  • the introduced double-stranded DNA fragments are provided in sufficient quantity to modify RNA molecule(s) in the cell but do not persist after a contemplated period of time has passed or after one or more cell divisions.
  • Methods for direct DNA transfer in plants are known by the skilled artisan (see for instance Davey et al. Plant Mol Biol. 1989 Sep;13(3):273-85.)
  • an RNA polynucleotide encoding the RNA targeting protein is introduced into the plant cell, which is then translated and processed by the host cell generating the protein in sufficient quantity to modify the RNA molecule(s) cell (in the presence of at least one guide RNA) but which does not persist after a contemplated period of time has passed or after one or more cell divisions.
  • Methods for introducing mRNA to plant protoplasts for transient expression are known by the skilled artisan (see for instance in Gallie, Plant Cell Reports (1993), 13; 119-122). Combinations of the different methods described above are also envisaged.
  • RNA targeting CRISPR components Delivery of RNA targeting CRISPR components to the plant cell
  • RNA targeting CRISPR system it is of interest to deliver one or more components of the RNA targeting CRISPR system directly to the plant cell. This is of interest, inter alia, for the generation of non-transgenic plants (see below).
  • one or more of the RNA targeting components is prepared outside the plant or plant cell and delivered to the cell.
  • the RNA targeting protein is prepared in vitro prior to introduction to the plant cell.
  • RNA targeting protein can be prepared by various methods known by one of skill in the art and include recombinant production. After expression, the RNA targeting protein is isolated, refolded if needed, purified and optionally treated to remove any purification tags, such as a His-tag. Once crude, partially purified, or more completely purified RNA targeting protein is obtained, the protein may be introduced to the plant cell.
  • the RNA targeting protein is mixed with guide RNA targeting the RNA of interest to form a pre-assembled ribonucleoprotein
  • the individual components or pre-assembled ribonucleoprotein can be introduced into the plant cell via electroporation, by bombardment with RNA targeting -associated gene product coated particles, by chemical transfection or by some other means of transport across a cell membrane.
  • transfection of a plant protoplast with a pre-assembled CRISPR ribonucleoprotein 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 the plants.
  • the RNA targeting CRISPR system components are introduced into the plant cells using nanoparticles.
  • the components either as protein or nucleic acid or in a combination thereof, can be uploaded onto or packaged in nanoparticles and applied to the plants (such as for instance described in WO 2008042156 and US 20130185823).
  • embodiments of the invention comprise nanoparticles uploaded with or packed with DNA molecule(s) encoding the RNA targeting protein, DNA molecules encoding the guide RNA and/or isolated guide RNA as described in WO2015089419.
  • RNA targeting CRISPR system comprises cell penetrating peptides (CPP).
  • CPP cell penetrating peptides
  • the invention comprises compositions comprising a cell penetrating peptide linked to an RNA targeting protein.
  • an RNA targeting protein and/or guide RNA(s) is coupled to one or more CPPs to effectively transport them inside plant protoplasts (Ramakrishna (2014, Genome Res. 2014 Jun;24(6): 1020-7 for Cas9 in human cells).
  • the RNA targeting gene and/or guide RNA(s) are encoded by one or more circular or non-circular DNA molecule(s) which are coupled to one or more CPPs for plant protoplast delivery.
  • the plant protoplasts are then regenerated to plant cells and further to plants.
  • CPPs are generally described as short peptides of fewer than 35 amino acids either derived from proteins or from chimeric sequences which are capable of transporting biomolecules across cell membrane in a receptor independent manner.
  • CPP can be cationic peptides, peptides having hydrophobic sequences, amphipatic peptides, peptides having proline-rich and antimicrobial sequence, and chimeric or bipartite peptides (Pooga and Langel 2005).
  • CPPs are able to penetrate biological membranes and as such trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, and hence facilitate interaction of the biolomolecule with the target.
  • Examples of CPP include amongst others: Tat, a nuclear transcriptional activator protein required for viral replication by HIV typel, penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin ⁇ 3 signal peptide sequence; polyarginine peptide Args sequence, Guanine rich-molecular transporters, sweet arrow peptide, etc...
  • the target RNA i.e. the RNA of interest
  • the target RNA is the RNA to be targeted by the present invention leading to the recruitment to, and the binding of the RNA targeting protein at, the target site of interest on the target RNA.
  • the target RNA may be any suitable form of RNA. This may include, in some embodiments, mRNA.
  • the target RNA may include transfer RNA (tRNA) or ribosomal RNA (rRNA).
  • the target RNA may include interfering RNA (RNAi), microR A (miRNA), microswitches, microzymes, satellite RNAs and RNA viruses.
  • the target RNA may be located in the cytoplasm of the plant cell, or in the cell nucleus or in a plant cell organelle such as a mitochondrion, chloroplast or piastid.
  • the RNA targeting CRISPR system is used to cleave RNA or otherwise inhibit RNA expression.
  • RNA targeting CRISPR system for modulating plant gene expression via RNA modulation
  • the RNA targeting protein may also be used, together with a suitable guide RNA, to target gene expression, via control of RNA processing.
  • the control of RNA processing may include RNA processing reactions such as RNA splicing, including alternative splicing or specifically targeting certain splice variants or isoforms; viral replication (in particular of plant viruses, including virioids in plants and tRNA biosynthesis.
  • the RNA targeting protein in combination with a suitable guide RNA may also be used to control RNA activation (RNAa).
  • RNAa leads to the promotion of gene expression, so control of gene expression may be achieved that way through di sruption or reduction of RNAa and thus less prom otion of gene expression.
  • the RNA targeting effector protein of the invention can further be used for antiviral activity in plants, in particular against RNA viruses.
  • the effector protein can be targeted to the viral RNA using a suitable guide RNA selective for a selected viral RNA sequence.
  • the effector protein may be an active nuclease that cleaves RNA, such as single stranded RNA. provided is therefore the use of an RNA targeting effector protein of the invention as an antiviral agent.
  • viruses that can be counteracted in this way include, but are not limited to, Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic vims (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PYX)
  • TMV Tobacco mosaic virus
  • TSWV Tomato spotted wilt virus
  • CMV Cucumber mosaic vims
  • PVY Potato virus Y
  • CaMV Cauliflower mosaic virus
  • PV Plum pox virus
  • BMV Brome mosaic virus
  • PYX Potato virus X
  • regulated control of gene expression through regulated cleavage of mRNA. This can be achieved by placing elements of the RNA targeting under the control of regulated promoters as described herein.
  • RNA targeting CRISPR system Use of the RNA targeting CRISPR system to restore the functionality of tRNA molecules.
  • RNA editing in plant mitochondria and chloroplasts that alters mRNA sequences to code for different proteins than the DNA.
  • the elements of the RNA targeting CRISPR system specifically targetting mitochondrial and chloroplast mRNA can be introduced in a plant or plant ceil to express different proteins in such plant cell organelles mimicking the processes occuring in vivo.
  • RNA targeting CRISPR system has uses similar to RNA inhibition or RNA interference, thus can also be substituted for such methods.
  • the methods of the present invention include the use of the RNA targeting CRISPR as a substitute for e.g. an interfering ribonucleic acid (such as an siRNA or shRNA or a dsRNA). Examples of inhibition of RNA expression in plants, algae or fungi as an alternative of targeted gene modification are described herein further.
  • control over interfering RNA or miRNA may help reduce off-target effects (OTE) seen with those approaches by reducing the longevity of the interfering RNA or miRNA in vivo or in vitro.
  • the target RNA may include interfering RNA, i.e. RNA involved in an RNA interference pathway, such as shRNA, siRNA and so forth.
  • the target RNA may include microRNA (miRNA) or double stranded RNA (dsRNA).
  • RNA targeting protein and suitable guide RNA(s) are selectively expressed (for example spatially or temporally under the control of a regulated promoter, for example a tissue- or cell cycle-specific promoter and/or enhancer) this can be used to 'protect' the cells or systems ⁇ in vivo or in vitro) from RNAi in those cells.
  • a regulated promoter for example a tissue- or cell cycle-specific promoter and/or enhancer
  • the RNA targeting protein may be used to control or bind to molecules comprising or consisting of RNA, such as ribozymes, ribosomes or riboswitches.
  • the guide RNA can recruit the RNA targeting protein to these molecules so that the RNA targeting protein is able to bind to them.
  • RNA targeting CRISPR system of the invention can be applied in areas of in- planta RNAi technologies, without undue experimentation, from this disclosure, including insect pest management, plant disease management and management of herbicide resistance, as well as in plant assay and for other applications (see, for instance Kim et al., in 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, inPest Management Science, Vol 70(9), pp 1351-1357), because the present application provides the foundation for informed engineering of the system.
  • Riboswitches are regulatory segments of messenger RNA that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules.
  • a particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene.
  • control of riboswitch activity is envisaged through the use of the RNA targeting protein in combination with a suitable guide RNA to target the riboswitch. This may be through cleavage of, or binding to, the riboswitch. In particular embodiments, reduction of riboswitch activity is envisaged.
  • TPP thiamin pyrophosphate
  • TPP riboswitches are also found in certain fungi, such as in 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)
  • uORF Upstream Open Reading Frame
  • the RNA targeting CRISPR system described herein may be used to manipulate the endogenous riboswitch activity in plants, algae or fungi and as such alter the expression of downstream genes controlled by it.
  • the RNA targeting CRISP system may be used in assaying riboswitch function in vivo or in vitro and in studying its relevance for the metabolic network.
  • the RNA targeting CRISPR system may potentially be used for engineering of riboswitches as metabolite sensors in plants and platforms for gene control.
  • RNA targeting CRISPR system Screens for plants, algae or fungi
  • RNAi screens Identifying gene products whose knockdown is associated with phenotypic changes, biological pathways can be interrogated and the constituent parts identified, via RNAi screens.
  • control may also be exerted over or during these screens by use of the Guide 29 or Guide 30 protein and suitable guide RNA described herein to remove or reduce the activity of the RNAi in the screen and thus reinstate the activity of the (previously interfered with) gene product (by removing or reducing the interference/repression).
  • RNA targeting proteins for visualization of RNA molecules in vivo and in vitro
  • the invention provides a nucleic acid binding system.
  • In situ hybridization of RNA with complementary probes is a powerful technique.
  • fluorescent DNA oligonucleotides are used to detect nucleic acids by hybridization.
  • Increased efficiency has been attained by certain modifications, such as locked nucleic acids (LNAs), but there remains a need for efficient and versatile alternatives.
  • LNAs locked nucleic acids
  • labelled elements of the RNA targeting system can be used as an alternative for efficient and adaptable system for in situ hybridization
  • biofuel is an alternative fuel made from plant and plant- derived resources. Renewable biofuels can be extracted from organic matter whose energy has been obtained through a process of carbon fixation or are made through the use or conversion of biomass. This biomass can be used directly for biofuels or can be converted to convenient energy containing substances by thermal conversion, chemical conversion, and biochemical conversion. This biomass conversion can result in fuel in solid, liquid, or gas form.
  • biofuels There are two types of biofuels: bioethanol and biodiesel.
  • Bioethanol is mainly produced by the sugar fermentation process of cellulose (starch), which is mostly derived from maize and sugar cane.
  • Biodiesel on the other hand is mainly produced from oil crops such as rapeseed, palm, and soybean. Biofuels are used mainly for transportation.
  • the methods using the RNA targeting CRISPR system as described herein are used to alter the properties of the cell wall in order to facilitate access by key hydrolysing agents for a more efficient release of sugars for fermentation.
  • the biosynthesis of cellulose and/or lignin are modified.
  • Cellulose is the major component of the cell wall.
  • the biosynthesis of cellulose and lignin are co-regulated. By reducing the proportion of lignin in a plant the proportion of cellulose can be increased.
  • the methods described herein are used to downregulate lignin biosynthesis in the plant so as to increase fermentable carbohydrates.
  • the methods described herein are used to downregulate 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) as disclosed in WO 2008064289 A2.
  • C3H 4-coumarate 3 -hydroxylase
  • the methods described herein are used to produce plant mass that produces lower levels of acetic acid during fermentation (see also WO 2010096488). Modifying yeast for Biofuel production
  • RNA targeting enzymes are used for bioethanol production by recombinant micro-organisms.
  • RNA targeting enzymes can be used to engineer micro-organisms, such as yeast, to generate biofuel or biopolymers from fermentable sugars and optionally to be able to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars.
  • the invention provides methods whereby the RNA targeting CRISPR complex is used to modify the expression of endogenous genes required for biofuel production and/or to modify endogenous genes why may interfere with the biofuel synthesis.
  • the methods involve stimulating the expression in a micro-organism such as a yeast of one or more nucleotide sequence encoding enzymes involved in the conversion of pyruvate to ethanol or another product of interest.
  • a micro-organism such as a yeast of one or more nucleotide sequence encoding enzymes involved in the conversion of pyruvate to ethanol or another product of interest.
  • the methods ensure the stimulation of expression of one or more enzymes which allows the micro-organism to degrade cellulose, such as a cellulase.
  • the RNA targeting CRISPR complex is used to suppress endogenous metabolic pathways which compete with the biofuel production pathway.
  • Transgenic algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.
  • alcohols especially methanol and ethanol
  • US 8945839 describes a method for engineering Micro- Algae (Chlamydomonas reinhardtii cells) species) using Cas9.
  • the methods of the RNA targeting CRISPR system described herein can be applied on Chlamydomonas species and other algae.
  • the RNA targeting effetor protein and guide RNA are introduced in algae expressed using a vector that expresses the RNA targeting effector protein under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2 -tubulin.
  • Guide RNA will be delivered using a vector containing T7 promoter.
  • in vitro transcribed guide RNA can be delivered to algae cells. Electroporation protocol follows standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.
  • present invention can be used as a therapy for virus removal in plant systems as it is able to cleave viral RNA.
  • Previous studies in human systems have demonstrated the success of utilizing CRISPR in targeting the single strand RNA virus, hepatitis C (A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods may also be adapted for using the RNA targeting CRISPR system in plants.
  • the present invention also provides plants and yeast cells obtainable and obtained by the methods provided herein.
  • the improved plants obtained by the methods described herein may be useful in food or feed production through the modified expression of genes which, for instance ensure tolerance to plant pests, herbicides, drought, low or high temperatures, excessive water, etc.
  • the improved plants obtained by the methods described herein, especially crops and algae may be useful in food or feed production through expression of, for instance, higher protein, carbohydrate, nutrient or vitamin levels than would normally be seen in the wildtype.
  • improved plants, especially pulses and tubers are preferred.
  • Improved algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.
  • alcohols especially methanol and ethanol
  • Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Plant parts as envisaged herein may be viable, nonviable, regeneratable, and/or non- regeneratable.
  • plant cells and plants generated according to the methods of the invention are also included within the scope of the present invention.
  • Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the genetic modification, which are produced by traditional breeding methods are also included within the scope of the present invention.
  • Such plants may contain a heterologous or foreign DNA sequence inserted at or instead of a target sequence.
  • such plants may contain only an alteration (mutation, deletion, insertion, substitution) in one or more nucleotides. As such, such plants will only be different from their progenitor plants by the presence of the particular modification.
  • a Casl3c system is used to engineer pathogen resistant plants, for example by creating resistance against diseases caused by bacteria, fungi or viruses.
  • pathogen resistance can be accomplished by engineering crops to produce a Casl 3c system that wil be ingested by an insect pest, leading to mortality.
  • a Casl3c system is used to engineer abiotic stress tolerance.
  • a Casl3c system is used to engineer drought stress tolerance or salt stress tolerance, or cold or heat stress tolerance. Younis et al. 2014, Int. J. Biol. Sci.
  • Some non-limiting target crops include Arabidops Zea mays is thaliana, Oryza sativa L, Prunus domestica L., Gossypium hirsutum, Nicotiana rustica, Zea mays, Medicago sativa, Nicotiana benthamiana and Arabidopsis thaliana
  • a Casl3c system is used for management of crop pests.
  • a Casl 3c system operable in a crop pest can be expressed from a plant host or transferred directly to the target, for example using a viral vector.
  • the invention provides a method of efficiently producing homozygous organisms from a heterozygous non-human starting organism.
  • the invention is used in plant breeding.
  • the invention is used in animal breeding.
  • a homozygous organism such as a plant or animal is made by preventing or suppressing recombination by interfering with at least one target gene involved in double strand breaks, chromosome pairing and/or strand exchange.
  • the invention provides a system for specific delivery of functional components to the RNA environment. This can be ensured using the CRISPR systems comprising the RNA targeting effector proteins of the present invention which allow specific targeting of different components to RNA. More particularly such components include activators or repressors, such as activators or repressors of RNA translation, degradation, etc. Applications of this system are described elsewhere herein.
  • the invention provides non-naturally occurring or engineered composition comprising a guide RNA comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the guide RNA is modified by the insertion of one or more distinct RNA sequence(s) that bind an adaptor protein.
  • the RNA sequences may bind to two or more adaptor proteins (e.g. aptamers), and wherein each adaptor protein is associated with one or more functional domains.
  • the guide RNAs of the Casl3c enzymes described herein are shown to be amenable to modification of the guide sequence.
  • the guide RNA is modified by the insertion of distinct RNA sequence(s) 5' of the direct repeat, within the direct repeat, or 3' of the guide sequence.
  • the functional domains can be same or different, e.g., two of the same or two different activators or repressors.
  • the invention provides a herein-discussed composition, wherein the one or more functional domains are attached to the RNA targeting enzyme so that upon binding to the target RNA the functional domain is in a spatial orientation allowing for the functional domain to function in its attributed function;
  • the invention provides a herein-discussed composition, wherein the composition comprises a CRISPR-Cas complex having at least three functional domains, at least one of which is associated with the RNA targeting enzyme and at least two of which are associated with the gRNA.
  • the invention provides non-naturally occurring or engineered CRISPR-Cas complex composition
  • the guide RNA as herein-discussed and a CRISPR enzyme which is an RNA targeting enzyme, wherein optionally the RNA targeting enzyme comprises at least one mutation, such that the RNA targeting enzyme has no more than 5% of the nuclease activity of the enzyme not having the at least one mutation, and optionally one or more comprising at least one or more nuclear localization sequences.
  • the guide RNA is additionally or alternatively modified so as to still ensure binding of the RNA targeting enzyme but to prevent cleavage by the RNA targeting enzyme (as detailed elsewhere herein).
  • the RNA targeting enzyme is a Casl3c enzyme which has a diminished nuclease activity of at least 97%, or 100% as compared with the Casl3c enzyme not having the at least one mutation.
  • the invention provides a herein-discussed composition, wherein the Casl3c enzyme comprises two or more mutations.
  • the mutations may be selected from mutations of one or more of the following amino acid residues: R597, H602, R1278, and H1283, such as for instance one or more of the following mutations: R597A, H602A, R1278A, and H1283A, according to Leptotrichia shahii Casl3c protein or a corresponding position in an ortholog.
  • an RNA targeting system comprising two or more functional domains.
  • the two or more functional domains are heterologous functional domain.
  • the system comprises an adaptor protein which is a fusion protein comprising a functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain.
  • the linker includes a GlySer linker.
  • one or more functional domains are attached to the RNA effector protein by way of a linker, optionally a GlySer linker.
  • the one or more functional domains are attached to the RNA targeting enzyme through one or both of the HEPN domains.
  • the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the adaptor protein or the RNA targeting enzume is a domain capable of activating or repressing RNA translation.
  • the invention provides a herein-discussed composition, wherein at least one of the one or more functional domains associated with the adaptor protein have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release 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 light inducibility.
  • the invention provides a herein-discussed composition comprising an aptamer sequence.
  • the aptamer sequence is two or more aptamer sequences specific to the same adaptor protein.
  • the invention provides a herein- discussed composition, wherein the aptamer sequence is two or more aptamer sequences specific to different adaptor protein.
  • the invention provides a herein-discussed composition, wherein the adaptor protein comprises MS2, PP7, Qp, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ )5, ( ⁇ Cb8r, ( 3 ⁇ 412r, (
  • the aptamer is selected from a binding protein specifically binding any one of the adaptor proteins listed above.
  • the invention provides a herein-discussed composition, wherein the cell is a eukaryotic cell.
  • the invention provides a herein-discussed composition, wherein the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell, whereby the mammalian cell is optionally a mouse cell.
  • the invention provides a herein-discussed composition, wherein the mammalian cell is a human cell.
  • the invention provides a herein above-discussed composition wherein there is more than one gRNA, and the gRNAs target different sequences whereby when the composition is employed, there is multiplexing.
  • the invention provides a composition wherein there is more than one gRNA modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins.
  • the invention provides a herein-discussed composition wherein one or more adaptor proteins associated with one or more functional domains is present and bound to the distinct RNA sequence(s) inserted into the guide RNA(s).
  • the invention provides a herein-discussed composition wherein the guide RNA is modified to have at least one non-coding functional loop; e.g., wherein the at least one non-coding functional loop is repressive; for instance, wherein at least one non-coding functional loop comprises Alu.
  • the invention provides a method for modifying gene expression comprising the administration to a host or expression in a host in vivo of one or more of the compositions as herein-discussed.
  • the invention provides a herein-discussed method comprising the delivery of the composition or nucleic acid molecule(s) coding therefor, wherein said nucleic acid molecule(s) are operatively linked to regulatory sequence(s) and expressed in vivo.
  • the invention provides a herein-discussed method wherein the expression in vivo is via a lentivirus, an adenovirus, or an AAV.
  • the invention provides a mammalian cell line of cells as herein-discussed, wherein the cell line is, optionally, a human cell line or a mouse cell line.
  • the invention provides a transgenic mammalian model, optionally a mouse, wherein the model has been transformed with a herein-discussed composition or is a progeny of said transformant.
  • the invention provides a nucleic acid molecule(s) encoding guide RNA or the RNA targeting CRISPR-Cas complex or the composition as herein-discussed.
  • the invention provides a vector comprising: a nucleic acid molecule encoding a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the direct repeat of the gRNA is modified by the insertion of distinct RNA sequence(s) that bind(s) to two or more adaptor proteins, and wherein each adaptor protein is associated with one or more functional domains; or, wherein the gRNA is modified to have at least one non-coding functional loop.
  • gRNA guide RNA
  • the invention provides vector(s) comprising nucleic acid molecule(s) encoding: non-naturally occurring or engineered CRISPR-Cas complex composition comprising the gRNA herein-discussed, and an RNA targeting enzyme, wherein optionally the RNA targeting enzyme comprises at least one mutation, such that the RNA targeting enzyme has no more than 5% of the nuclease activity of the RNA targeting enzyme not having the at least one mutation, and optionally one or more comprising at least one or more nuclear localization sequences.
  • a vector can further comprise regulatory element(s) operable in a eukaryotic cell operably linked to the nucleic acid molecule encoding the guide RNA (gRNA) and/or the nucleic acid molecule encoding the RNA targeting enzyme and/or the optional nuclear localization sequence(s).
  • regulatory element(s) operable in a eukaryotic cell operably linked to the nucleic acid molecule encoding the guide RNA (gRNA) and/or the nucleic acid molecule encoding the RNA targeting enzyme and/or the optional nuclear localization sequence(s).
  • the invention provides a kit comprising one or more of the components described hereinabove.
  • the kit comprises a vector system as described above and instructions for using the kit.
  • the invention provides a method of 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) comprising the cell line of as herein-discussed or cells of the model herein- discussed containing or expressing the RNA targeting enzyme and introducing a composition as herein-discussed into cells of the cell line or model, whereby the gRNA includes either an activator or a repressor, and monitoring for GOF or LOF respectively as to those cells as to which the introduced gRNA includes an activator or as to those cells as to which the introduced gRNA includes a repressor.
  • GEF gain of function
  • LEF loss of function
  • non-coding RNAs or potential regulatory regions e.g. enhancers, repressors
  • the invention provides a library of non-naturally occurring or engineered compositions, each comprising a 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 no more than 5% of the nuclease activity of the RNA targeting enzyme not having the at least one mutation, wherein the gRNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein the composition comprises one or more or two or more adaptor proteins, wherein the each protein is associated with one or more functional domains, and wherein the gRNAs comprise a genome wide library comprising a plurality of RNA targeting guide RNAs (gRNAs).
  • gRNAs RNA targeting CRISPR guide RNA
  • the invention provides a library as herein-discussed, wherein the RNA targeting RNA targeting enzyme has a diminished nuclease activity of at least 97%, or 100%) as compare with the RNA targeting enzyme not having the at least one mutation.
  • the invention provides a library as herein-discussed, wherein the adaptor protein is a fusion protein comprising the functional domain.
  • the invention provides a library as herein discussed, wherein the gRNA is not modified by the insertion of distinct RNA sequence(s) that bind to the one or two or more adaptor proteins.
  • the invention provides a library as herein discussed, wherein the one or two or more functional domains are associated with the RNA targeting enzyme.
  • the invention provides a library as herein discussed, wherein the cell population of cells is a population of eukaryotic cells.
  • the invention provides a library as herein discussed, wherein the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell.
  • the invention provides a library as herein discussed, wherein the mammalian cell is a human cell.
  • the invention provides a library as herein discussed, wherein the population of cells is a population of embryonic stem (ES) cells.
  • ES embryonic stem
  • the invention provides a library as herein discussed, wherein the targeting is of about 100 or more RNA sequences. In an aspect the invention provides a library as herein discussed, wherein the targeting is of about 1000 or more RNA sequences. In an aspect the invention provides a library as herein discussed, wherein the targeting is of about 20,000 or more sequences. In an aspect the invention provides a library as herein discussed, wherein the targeting is of the entire transcriptome. In an aspect the invention provides a library as herein discussed, wherein the targeting is of a panel of target sequences focused on a relevant or desirable pathway. In an aspect the invention provides a library as herein discussed, wherein the pathway is an immune pathway. In an aspect the invention provides a library as herein discussed, wherein the pathway is a cell division pathway.
  • the invention provides a method of generating a model eukaryotic cell comprising a gene with modified expression.
  • a disease gene is any gene associated an increase in the risk of having or developing a disease.
  • the method comprises (a) introducing one or more vectors encoding the components of the system described herein above into a eukaryotic cell, and (b) allowing a CRISPR complex to bind to a target polynucleotide so as to modify expression of a gene, thereby generating a model eukaryotic cell comprising modified gene expression.
  • the structural information provided herein allows for interrogation of guide RNA interaction with the target RNA and the RNA targeting enzyme permitting engineering or alteration of guide RNA structure to optimize functionality of the entire RNA targeting CRISPR- Cas system.
  • the guide RNA may be extended, without colliding with the RNA targeting protein by the insertion of adaptor proteins that can bind to RNA. These adaptor proteins can further recruit effector proteins or fusions which comprise one or more functional domains.
  • An aspect of the invention is that the above elements are comprised in a single composition or comprised in individual compositions. These compositions may advantageously be applied to a host to elicit a functional effect on the genomic level.
  • modifications to the guide RNA which allow for binding of the adapter + functional domain but not proper positioning of the adapter + functional domain (e.g. due to steric hindrance within the three dimensial structure of the CRISPR complex) are modifications which are not intended.
  • the one or more modified guide RNA may be modified, by introduction of a distinct RNA sequence(s) 5' of the direct repeat, within the direct repeat, or 3' of the guide sequence.
  • the modified guide RNA, the inactivated RNA targeting enzyme (with or without functional domains), and the binding protein with one or more functional domains may each individually be comprised in a composition and administered to a host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host. Administration to a host may be performed via viral vectors known to the skilled person or described herein for delivery to a host (e.g. lentiviral vector, adenoviral vector, AAV vector). As explained herein, use of different selection markers (e.g. for lentiviral gRNA selection) and concentration of gRNA (e.g. dependent on whether multiple gRNAs are used) may be advantageous for eliciting an improved effect.
  • compositions can advantageously and specifically target single or multiple loci with the same or different functional domains to elicit one or more genomic events.
  • the compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g. gene activation of lincRNA and indentification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes).
  • the current invention comprehends the use of the compositions of the current invention to establish and utilize conditional or inducible CRISPR RNA targeting events.
  • CRISPR RNA targeting events See, e.g., Piatt 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 (PCT/US2013/074667), which are not believed prior to the present invention or application).
  • the target cell comprises RNA targeting CRISRP enzyme conditionally or inducibly (e.g.
  • the adapter protein conditionally or inducibly and, on expression of a vector introduced into the target cell, the vector expresses that which induces or gives rise to the condition of s RNA targeting enzyme expression and/or adaptor expression in the target cell.
  • the adaptor protein may be provided as a conditional or inducible element with a conditional or inducible s RNA targeting enzyme to provide an effective model for screening purposes, which advantageously only requires minimal design and administration of specific gRNAs for a broad number of applications.
  • Guide RNA according to the invention comprising a dead guide sequence
  • the invention provides guide sequences which are modified in a manner which allows for formation of the CRISPR complex and successful binding to the target, while at the same time, not allowing for successful nuclease activity (i.e. without nuclease activity / without indel activity).
  • modified guide sequences are referred to as "dead guides” or “dead guide sequences”.
  • dead guides or dead guide sequences can be thought of as catalytically inactive or conformationally inactive with regard to nuclease activity. Indeed, dead guide sequences may not sufficiently engage in productive base pairing with respect to the ability to promote catalytic activity or to distinguish on-target and off-target binding activity.
  • the assay involves synthesizing a CRISPR target RNA and guide RNAs comprising mismatches with the target RNA, combining these with the RNA targeting enzyme and analyzing cleavage based on gels based on the presence of bands generated by cleavage products, and quantifying cleavage based upon relative band intensities.
  • the invention provides a non-naturally occurring or engineered composition RNA targeting CRISPR-Cas system comprising a functional RNA targeting as described herein, and guide RNA (gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the RNA targeting CRISPR-Cas system is directed to a genomic locus of interest in a cell without detectable RNA cleavage activity of a non-mutant RNA targeting enzyme of the system.
  • gRNA guide RNA
  • any of the gRNAs according to the invention as described herein elsewhere may be used as dead gRNAs / gRNAs comprising a dead guide sequence as described herein below. Any of the methods, products, compositions and uses as described herein elsewhere is equally applicable with the dead gRNAs / gRNAs comprising a dead guide sequence as further detailed below.
  • the ability of a dead guide sequence to direct sequence-specific binding of a CRISPR complex to an RNA target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the dead guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target RNA polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the dead guide sequence to be tested and a control guide sequence different from the test dead guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a dead guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Dead guide sequences are typically shorter than respective guide sequences which result in active RNA cleavage.
  • dead guides are 5%, 10%, 20%, 30%), 40%), 50%), shorter than respective guides directed to the same.
  • one aspect of gRNA - RNA targeting specificity is the direct repeat sequence, which is to be appropriately linked to such guides. In particular, this implies that the direct repeat sequences are designed dependent on the origin of the RNA targeting enzyme. Thus, structural data available for validated dead guide sequences may be used for designing Casl3c specific equivalents.
  • the dead guide herein may be appropriately modified in length and sequence to reflect such Casl3c specific equivalents, allowing for formation of the CRISPR complex and successful binding to the target RNA, while at the same time, not allowing for successful nuclease activity.
  • dead guides in the context herein as well as the state of the art provides a surprising and unexpected platform for network biology and/or systems biology in both in vitro, ex vivo, and in vivo applications, allowing for multiplex gene targeting, and in particular bidirectional multiplex gene targeting.
  • addressing multiple targets Prior to the use of dead guides, addressing multiple targets has been challenging and in some cases not possible.
  • multiple targets, and thus multiple activities may be addressed, for example, in the same cell, in the same animal, or in the same patient. Such multiplexing may occur at the same time or staggered for a desired timeframe.
  • the dead guides allow to use gRNA as a means for gene targeting, without the consequence of nuclease activity, while at the same time providing directed means for activation or repression.
  • Guide RNA comprising a dead guide may be modified to further include elements in a manner which allow for activation or repression of gene activity, in particular protein adaptors (e.g. aptamers) as described herein elsewhere allowing for functional placement of gene effectors (e.g. activators or repressors of gene activity).
  • protein adaptors e.g. aptamers
  • gene effectors e.g. activators or repressors of gene activity.
  • One example is the incorporation of aptamers, as explained herein and in the state of the art.
  • gRNA By engineering the gRNA comprising a dead guide to incorporate protein-interacting aptamers (Konermann et al., "Genome-scale transcription activation by an engineered CRISPR-Cas9 complex," doi: 10.1038/naturel4136, incorporated herein by reference), one may assemble multiple distinct effector domains. Such may be modeled after natural processes.
  • a gRNA of the invention which comprises a dead guide, wherein the gRNA further comprises modifications which provide for gene activation or repression, as described herein.
  • the dead gRNA may comprise one or more aptamers.
  • the aptamers may be specific to gene effectors, gene activators or gene repressors.
  • the aptamers may be specific to a protein which in turn is specific to and recruits / binds a specific gene effector, gene activator or gene repressor. If there are multiple sites for activator or repressor recruitment, it is preferred that the sites are specific to either activators or repressors.
  • the sites may be specific to the same activators or same repressors.
  • the sites may also be specific to different activators or different repressors.
  • the effectors, activators, repressors may be present in the form of fusion proteins.
  • the invention provides a method of selecting a dead guide RNA targeting sequence for directing a functionalized CRISPR system to a gene locus in an organism, which comprises: a) locating one or more CRISPR motifs in the gene locus; b) analyzing the 20 nt sequence downstream of each CRISPR motif by: i) determining the GC content of the sequence; and ii) determining whether there are off-target matches of the first 15 nt of the sequence in the genome of the organism; c) selecting the sequence for use in a guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In an embodiment, the sequence is selected if the GC content is 50% or less.
  • the sequence is selected if the GC content is 40% or less. In an embodiment, the sequence is selected if the GC content is 30% or less. In an embodiment, two or more sequences are analyzed and the sequence having the lowest GC content is selected. In an embodiment, off-target matches are determined in regulatory sequences of the organism. In an embodiment, the gene locus is a regulatory region. An aspect provides a dead guide RNA comprising the targeting sequence selected according to the aforementioned methods.
  • the invention provides a dead guide RNA for targeting a functionalized CRISPR system to a gene locus in an organism.
  • the dead guide RNA comprises a targeting sequence wherein the CG content of the target sequence is 70% or less, and the first 15 nt of the targeting sequence does not match an off-target sequence downstream from a CRISPR motif in the regulatory sequence of another gene locus in the organism.
  • the GC content of the targeting sequence 60% or less, 55% or less, 50% or less, 45%) or less, 40% or less, 35% or less or 30% or less.
  • the GC content of the targeting sequence is from 70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%.
  • the targeting sequence has the lowest CG content among potential targeting sequences of the locus.
  • the first 15 nt of the dead guide match the target sequence.
  • first 14 nt of the dead guide match the target sequence.
  • the first 13 nt of the dead guide match the target sequence.
  • first 12 nt of the dead guide match the target sequence.
  • first 11 nt of the dead guide match the target sequence.
  • the first 10 nt of the dead guide match the target sequence.
  • the first 15 nt of the dead guide does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus.
  • the first 14 nt, or the first 13 nt of the dead guide, or the first 12 nt of the guide, or the first 11 nt of the dead guide, or the first 10 nt of the dead guide does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus.
  • the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt of the dead guide do not match an off-target sequence downstream from a CRISPR motif in the genome.
  • the dead guide RNA includes additional nucleotides at the 3'- end that do not match the target sequence.
  • a dead guide RNA that includes the first 20-28 nt, downstream of a CRISPR motif can be extended in length at the 3' end.
  • the invention provides a nucleic acid binding system.
  • In situ hybridization of RNA with complementary probes is a powerful technique.
  • fluorescent DNA oligonucleotides are used to detect nucleic acids by hybridization.
  • Increased efficiency has been attained by certain modifications, such as locked nucleic acids (LNAs), but there remains a need for efficient and versatile alternatives.
  • LNAs locked nucleic acids
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non- limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina,
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10 - 30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.”
  • Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • promoters e.g., promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • IRES internal ribosomal entry sites
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue- specific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a vector comprises one or more pol III promoter (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.
  • pol III promoters include, but are not limited to, U6 and HI promoters.
  • 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 ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5' segment in 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 ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • WPRE WPRE
  • CMV enhancers the R-U5' segment in LTR of HTLV-I
  • SV40 enhancer SV40 enhancer
  • the intron sequence between exons 2 and 3 of rabbit ⁇ -globin Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.
  • the CRISPR system as provided herein can make use of a crRNA or analogous polynucleotide comprising a guide sequence, wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA, and/or wherein the polynucleotide comprises one or more nucleotide analogs.
  • the sequence can comprise any structure, including but not limited to a structure of a native crRNA, such as a bulge, a hairpin or a stem loop structure.
  • the polynucleotide comprising the guide sequence forms a duplex with a second polynucleotide sequence which can be an RNA or a DNA sequence.
  • guides of the invention comprise non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemically modifications.
  • Non-naturally occurring nucleic acids can include, for example, mixtures of naturally and non-naturally occurring nucleotides.
  • Non-naturally occurring nucleotides and/or nucleotide analogs may be modified at the ribose, phosphate, and/or base moiety.
  • a guide nucleic acid comprises ribonucleotides and non-ribonucleotides.
  • a guide comprises one or more ribonucleotides and one or more deoxyribonucleotides.
  • the guide comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, or bridged nucleic acids (BNA).
  • LNA locked nucleic acid
  • modified nucleotides include 2'-0-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs.
  • modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine ( ⁇ ), N ⁇ methylpseudouridine (me lv P), 5-methoxyuridine(5moU), inosine, 7- methylguanosine.
  • Examples of guide RNA chemical modifications include, without limitation, incorporation of 2'-0-methyl (M), 2'-0-methyl 3 'phosphorothioate (MS), ⁇ -constrained ethyl (cEt), or 2'-0-methyl 3'thioPACE (MSP) at one or more terminal nucleotides.
  • M 2'-0-methyl
  • MS 2'-0-methyl 3 'phosphorothioate
  • cEt ⁇ -constrained ethyl
  • MSP 2'-0-methyl 3'thioPACE
  • the 5' and/or 3' end of a guide RNA is modified by a variety of functional moieties including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags. (See Kelly et al., 2016, J. Biotech. 233 :74-83).
  • a guide comprises ribonucleotides in a region that binds to a target DNA and one or more deoxyribonucletides and/or nucleotide analogs in a region that binds to Cas9, Cpfl, or C2cl .
  • deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide structures, such as, without limitation, 5' and/or 3' end, stem-loop regions, and the seed region.
  • the modification is not in the 5'-handle of the stem-loop regions.
  • Chemical modification in the 5 '-handle of the stem-loop region of a guide may abolish its function (see Li, et al., Nature Biomedical Engineering, 2017, 1 :0066).
  • 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 a guide is chemically modified.
  • 3-5 nucleotides at either the 3' or the 5' end of a guide is chemically modified.
  • only minor modifications are introduced in the seed region, such as 2'-F modifications.
  • 2'-F modification is introduced at the 3' end of a guide.
  • three to five nucleotides at the 5' and/or the 3' end of the guide are chemicially modified with 2'-0-methyl (M), 2'-0-methyl-3'-phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-0-methyl-3'- thioPACE (MSP).
  • M 2'-0-methyl
  • MS 2'-0-methyl-3'-phosphorothioate
  • cEt S-constrained ethyl
  • MSP 2'-0-methyl-3'- thioPACE
  • all of the phosphodiester bonds of a guide are substituted with phosphorothioates (PS) for enhancing levels of gene disruption.
  • more than five nucleotides at the 5' and/or the 3' end of the guide are chemicially modified with 2'-0-Me, 2'-F or ⁇ -constrained ethyl(cEt).
  • Such chemically modified guide can mediate enhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS, E7110- E7111).
  • a guide is modified to comprise a chemical moiety at its 3' and/or 5' end.
  • moieties include, but are not limited to amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), or Rhodamine.
  • the chemical moiety is conjugated to the guide by a linker, such as an alkyl chain.
  • the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, protein, or nanoparticles.
  • Such chemically modified guide can be used to identify or enrich cells generically edited by a CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI: 10.7554)
  • the guide comprises a modified crRNA for Cpfl, having a 5 '-handle and a guide segment further comprising a seed region and a 3 '-terminus.
  • the modified guide can be used with a Cpfl of any one of Acidaminococcus sp. BV3L6 Cpfl (AsCpfl); Francisella tularensis subsp. Novicida Ul 12 Cpfl (FnCpfl); L.
  • bacterium MA2020 Cpfl Lb2Cpfl
  • Porphyromonas crevioricanis Cpfl PeCpfl
  • Porphyromonas macacae Cpfl PmCpfl
  • Candidatus Methanoplasma termitum Cpfl CtCpfl
  • Eubacterium eligens Cpfl EeCpfl
  • Moraxella bovoculi 237 Cpfl MbCpfl
  • Prevotella disiens Cpfl PdCpfl
  • LbCpfl L. bacterium D2006 Cpfl
  • the modification to the guide is a chemical modification, an insertion, a deletion or a split.
  • the chemical modification includes, but is not limited to, incorporation of 2'-0-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine ( ⁇ ), N ⁇ methylpseudouridine (me lv P), 5-methoxyuridine(5moU), inosine, 7-methylguanosine, 2'- 0-methyl-3'-phosphorothioate (MS), ⁇ -constrained ethyl(cEt), phosphorothioate (PS), or 2'-0- methyl-3'-thioPACE (MSP).
  • M 2'-0-methyl
  • 2-thiouridine analogs N6-methyladenosine analogs
  • 2'-fluoro analogs 2-aminopurine
  • the guide comprises one or more of 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 in the seed region are chemically modified. In certain embodiments, one or more nucleotides in the 3 '-terminus are chemically modified. In certain embodiments, none of the nucleotides in the 5 '-handle is chemically modified. In some embodiments, the chemical modification in the seed region is a minor modification, such as incorporation of a 2'-fluoro analog.
  • one nucleotide of the seed region is replaced with a 2'-fluoro analog.
  • 5 or 10 nucleotides in the 3 '-terminus are chemically modified. Such chemical modifications at the 3 '-terminus of the Cpfl CrRNA improve gene cutting efficiency (see Li, et al., Nature Biomedical Engineering, 2017, 1 :0066).
  • 5 nucleotides in the 3 '-terminus are replaced with 2'-fluoro analogues.
  • 10 nucleotides in the 3 '-terminus are replaced with 2'-fluoro analogues.
  • 5 nucleotides in the 3 '-terminus are replaced with 2'- O-methyl (M) analogs.
  • the loop of the 5'-handle of the guide is modified. In some embodiments, the loop of the 5 '-handle of the guide is modified to have a deletion, an insertion, a split, or chemical modifications. In certain embodiments, the loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence of UCUU, UUUU, UAUU, or UGUU.
  • the guide comprises portions that are chemically linked or conjugated via a non-phosphodiester bond.
  • the guide comprises, in non-limiting examples, a tracr sequence and a tracr mate sequence portion or a direct repeat and a targeting sequence portion that are chemically linked or conjugated via a non-nucleotide loop.
  • the portions are joined via a non-phosphodiester covalent linker.
  • covalent linker examples include but are not limited to a chemical moiety selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C-C bond forming groups such as Diels-Alder cyclo-addition pairs or ring-closing metathesis pairs, and Michael reaction pairs.
  • a chemical moiety selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phospho
  • portions of the guide are first synthesized using the standard phosphoramidite synthetic protocol (Herdewijn, P., ed., Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)).
  • the non-targeting guide portions can be functionalized to contain an appropriate functional group for ligation using the standard protocol known in the art (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, thio semicarbazide, thiol, maleimide, haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide.
  • Examples of chemical bonds include, but are not limited to, those based on carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C-C bond forming groups such as Diels-Alder cyclo-addition pairs or ring- closing metathesis pairs, and Michael reaction pairs.
  • one or more portions of a guide can be chemically synthesized.
  • the chemical synthesis uses automated, solid-phase oligonucleotide synthesis machines with 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or 2'-thionocarbamate (2'-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133 : 11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33 :985-989).
  • 2'-ACE 2'-acetoxyethyl orthoester
  • the guide portions can be covalently linked using various bioconjugation reactions, loops, bridges, and non-nucleotide links via modifications of sugar, internucleotide phosphodiester bonds, purine and pyrimidine residues.
  • the guide portions can be covalently linked using click chemistry.
  • guide portions can be covalently linked using a triazole linker.
  • guide portions can be covalently linked using Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and azide to yield a highly stable triazole linker (He et al., ChemBioChem (2015) 17: 1809-1812; WO 2016/186745).
  • guide portions are covalently linked by ligating a 5'-hexyne portion and a 3 '-azide portion.
  • either or both of the 5'-hexyne guide portion and a 3 '-azide guide portion can be protected with 2'-acetoxyethl orthoester (2' -ACE) group, which can be subsequently removed using Dharmacon protocol (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18).
  • 2' -ACE 2'-acetoxyethl orthoester
  • guide portions can be covalently linked via a linker (e.g., a non- nucleotide loop) that comprises a moiety such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye labeled RNAs, and non-naturally occurring nucleotide analogues.
  • a linker e.g., a non- nucleotide loop
  • a moiety such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye labeled RNAs, and non-naturally occurring nucleotide analogues.
  • suitable spacers for purposes of this invention include, but are not limited to, polyethers (e.g., polyethylene glycols, polyalcohols, polypropylene glycol or mixtures of ethylene and propylene glycols), polyamines group (e.g., spennine, spermidine and polymeric derivatives thereof), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof.
  • Suitable attachments include any moiety that can be added to the linker to add additional properties to the linker, such as but not limited to, fluorescent labels.
  • Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacyl glycerols and dialkyl glycerols, 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. The design of example linkers conjugating two RNA components are also described in WO 2004/015075.
  • the linker (e.g., a non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equivalent to about 0-16 nucleotides. In some embodiments, the linker has a length equivalent to about 0-8 nucleotides. In some embodiments, the linker has a length equivalent to about 0-4 nucleotides. In some embodiments, the linker has a length equivalent to about 2 nucleotides.
  • Example linker design is also described in WO2011/008730.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%), 99%), or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San
  • a guide sequence within a nucleic acid-targeting guide RNA
  • a guide sequence may direct sequence-specific binding of a nucleic acid -targeting complex to a target nucleic acid sequence
  • the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid -targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid -targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein.
  • preferential targeting e.g., cleavage
  • cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid -targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence, and hence a nucleic acid- targeting guide RNA may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence may be any RNA sequence.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomaal RNA (rRNA), transfer RNA (tRNA), micro- RNA (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 (IncRNA), and small cytoplasmatic RNA (scRNA).
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.
  • a nucleic acid-targeting guide RNA is selected to reduce the degree secondary structure within the RNA-targeting guide RNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal 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 example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence.
  • the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5') from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') from the guide sequence or spacer sequence.
  • 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.
  • the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides, preferably at least 18 nt, such at at least 19, 20, 21, 22, or more nt.
  • the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
  • Applicants also perform a challenge experiment to verify the RNA targeting and cleaving capability of a Casl3c. This experiment closely parallels similar work in E. coli for the heterologous expression of StCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)). Applicants introduce a plasmid containing both a PAM and a resistance gene into the heterologous E. coli, and then plate on the corresponding antibiotic. If there is RNA cleavage of the plasmid transcribed resistance gene, Applicants observe no viable colonies.
  • the assay is as follows for a DNA target, but may be adapted accordingly for an RNA target.
  • Two E.coli strains are used in this assay. One carries a plasmid that encodes the endogenous effector protein locus from the bacterial strain. The other strain carries an empty plasmid (e.g.pACYC184, control strain). All possible 7 or 8 bp PAM sequences are presented on an antibiotic resistance plasmid (pUC19 with ampicillin resistance gene). The PAM is located next to the sequence of proto-spacer 1 (the DNA target to the first spacer in the endogenous effector protein locus). Two PAM libraries were cloned.
  • One has a 8 random bp 5' of the proto-spacer (e.g. total of 65536 different PAM sequences complexity).
  • Plasmid DNA was used as template for PCR amplification and subsequent deep sequencing. Representation of all PAMs in the untransfomed libraries showed the expected representation of PAMs in transformed cells. Representation of all PAMs found in control strains showed the actual representation. Representation of all PAMs in test strain showed which PAMs are not recognized by the enzyme and comparison to the control strain allows extracting the sequence of the depleted PAM.
  • nucleic acid-targeting guide RNA For minimization of toxicity and off-target effect, it will be important to control the concentration of nucleic acid-targeting guide RNA delivered.
  • Optimal concentrations of nucleic acid -targeting guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. The concentration that gives the highest level of on-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery.
  • the nucleic acid-targeting system is derived advantageously from a Type VI CRISPR system.
  • one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous RNA-targeting system.
  • the Type VI RNA-targeting Cas enzyme is Casl3c.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • the Type VI protein such as Cas 13 referred to herein also encompasses a homologue or an orthologue of a Type VI protein such as Casl3c.
  • the terms "orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art.
  • a "homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related.
  • an "orthologue" of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related, or are only partially structurally related.
  • the homologue or orthologue of a Type VI protein such as Casl 3c as referred to herein has a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with a Type VI protein such as Cas 13c.
  • the homologue or orthologue of a Type VI protein such as Casl3c as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type Type VI protein such as Casl3c.
  • the Type VI RNA-targeting Cas protein may be a Casl3cortholog of an organism of a genus which includes but is not limited to Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. Species of organism of such a genus can be as otherwise herein discussed.
  • Some methods of identifying orthologs of CRISPR-Cas system enzymes may involve identifying tracr sequences in genomes of interest. Identification of tracr sequences may relate to the following steps: Search for the direct repeats or tracr mate sequences in a database to identify a CRISPR region comprising a CRISPR enzyme. Search for homologous sequences in the CRISPR region flanking the CRISPR enzyme in both the sense and antisense directions. Look for transcriptional terminators and secondary structures. Identify any sequence that is not a direct repeat or a tracr mate sequence but has more than 50% identity to the direct repeat or tracr mate sequence as a potential tracr sequence. Take the potential tracr sequence and analyze for transcriptional terminator sequences associated therewith.
  • chimeric enzymes may comprise fragments of CRISPR enzyme orthologs of an organism which includes but is not limited to Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
  • a chimeric enzyme can comprise a first fragment and a second fragment, and the fragments can be of CRISPR enzyme orthologs of organisms of genuses herein mentioned or of species herein mentioned; advantageously the fragments are from CRISPR enzyme orthologs of different species.
  • the Type VI RNA-targeting effector protein in particular the Casl3c protein as referred to herein also encompasses a functional variant of Casl3c or a homologue or an orthologue thereof.
  • a "functional variant" of a protein as used herein refers to a variant of such protein which retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc.

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Abstract

L'invention concerne des systèmes, des méthodes et des compositions pour le ciblage d'acides nucléiques. En particulier, l'invention concerne des systèmes de ciblage d'ARN d'origine non naturelle ou obtenus par génie génétique comprenant une nouvelle protéine effectrice CRISPR de ciblage de l'ARN et au moins un constituant de type acide nucléique de ciblage, tel qu'un ARN guide.
EP18824952.8A 2017-06-26 2018-06-26 Nouveaux orthologues de crispr de type vi et systèmes associés Pending EP3645728A4 (fr)

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