WO2019094969A1 - Compositions, systèmes, kits et procédés de modification d'arn - Google Patents

Compositions, systèmes, kits et procédés de modification d'arn Download PDF

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Publication number
WO2019094969A1
WO2019094969A1 PCT/US2018/060831 US2018060831W WO2019094969A1 WO 2019094969 A1 WO2019094969 A1 WO 2019094969A1 US 2018060831 W US2018060831 W US 2018060831W WO 2019094969 A1 WO2019094969 A1 WO 2019094969A1
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rna
mmc2
virus
cell
effector protein
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PCT/US2018/060831
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Simone M. MANTOVANI
Russell D. Monds
Matthew C. Lafave
Robert C. Brown
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Synthetic Genomics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR- Cas CRISPR-associated genes adaptive immune systems, which protect microbes from viruses and other invading nucleic acid through three steps: (i) adaptation, i.e., insertion of foreign nucleic acid segments (spacers) into the CRISPR array in between pairs of direct repeats (DRs), (ii) transcription and processing of the CRISPR array to produce mature CRISPR RNAs (crRNAs), and (iii) interference, whereby Cas enzymes are guided by the crRNAs to target and cleave cognate sequences in the respective invader genomes
  • DRs direct repeats
  • crRNAs mature CRISPR RNAs
  • interference whereby Cas enzymes are guided by the crRNAs to target and cleave cognate sequences in the respective invader genomes
  • the CRISPR-Cas systems of bacterial and archaeal adaptive immunity show an extreme diversity of protein composition and genomic loci architecture.
  • the CRISPR- Cas system has an extreme diversity of loci architecture.
  • the CRISPR-Cas systems are broadly divided into two classes, Class 1 with multi-subunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Within these two classes, CRISPR-Cas systems were further subdivided into types and subtypes according to the presence of distinct signature genes, protein sequence conservation, and organization of the respective genomic loci.
  • Class 2 CRISPR systems comprise type II, type V, and type VI characterized by the single-component effector 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.
  • dsDNA double-stranded DNA
  • type III and VI CRISPR-Cas systems target RNA.
  • the type VI systems catalyze exhibit non-specific degradation of proximal ssRNA upon activation.
  • Mmc2 Clustered Regularly Interspersed Short Palindromic Repeat CRISPR
  • the system includes a) an Mmc2 effector protein, or one or more nucleotide sequences encoding an Mmc2 effector protein in which one or more crRNAs form one or more complexes with the Mmc2 effector protein, and one or more crRNAs hybridize to the one or more target RNAs and b) one or more engineered or non-naturally occurring Type VI CRISPR-Cas polynucleotide sequences encoding or comprising one or more crRNAs in which one or more crRNAs is capable of hybridizing with one or more target RNAs.
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeat
  • one or more nucleotide sequences encoding a Mmc2 effector protein and/or one or more engineered or non-naturally occurring Type VI CRISPR-Cas polynucleotide sequences encoding or comprising one or more crRNAs are operably linked to one or more regulatory elements.
  • the regulatory elements are same. In some embodiments, the regulatory elements are different. In some embodiments, the regulatory elements are inducible.
  • the system includes one or more vectors having: a) a first regulatory element operably linked to a nucleotide sequence encoding an Mmc2 effector protein in which the crRNA and the Mmc2 effector protein are located on the same or different vectors of the system, and b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more Type VI CRISPR-Cas polynucleotide sequences comprising a crRNAs in which the crRNAs is capable of hybridizing with one or more target RNAs.
  • CRISPRvector system includes one or more vectors having: a) a first regulatory element operably linked to a nucleotide sequence encoding an Mmc2 effector protein in which the crRNA and the Mmc2 effector protein are located on the same or different vectors of the system, and b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more Type VI CRISPR-Cas
  • one or more crRNAs when transcribed, form one or more complexes with the Mmc2 effector protein, and one or more crRNAs hybridize to the one or more target RNAs.
  • one or more vector is a viral vector.
  • viral vectors include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
  • two or more crRNA hybridize to two or more different target RNA and the Mmc2 effector protein cleaves two or more target RNA hybridized to the crRNA. In some embodiments, two or more crRNA hybridize to different sites of the same target RNA and the Mmc2 effector cleaves the same target RNA at two or more sites.
  • the vector system or the Mmc2 CRISPR-Cas system further includes divalent metal ion. Non-limiting examples of divalent metal ion includes Mg2+, Mn2+, Cu2+, Fe2+, Zn2+, Co2+.
  • the concentration of the divalent metal ion is about O. lmM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, ImM, 1.25mM, 1.5mM, 1.6mM, 1.75mM, 2mM, 2.25mM, 2.5mM, 2.75mM, 3mM, 3.25mM, 3.5mM, 3.75mM, 4mM, 4.25mM, 4.5mM, 4.75mM, 5mM, or more.
  • the methods include delivering to the one or more target RNA a non-naturally occurring or engineered composition comprising: a) an Mmc2 effector protein, or one or more nucleotide sequences encoding an Mmc2 effector protein, and b) one or more engineered or non- naturally occurring Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs in which one or more crRNAs is capable of hybridizing with one or more target RNA.
  • the one or more crRNAs form one or more complexes with the Mmc2 effector protein, and one or more crRNAs sequences hybridize to the one or more target RNA.
  • RNA detection methods include: a) providing a sample comprising one or more RNA; b) contacting the one or more RNA with: ii) an Mmc2 effector protein, or one or more nucleotide sequences encoding an Mmc2 effector protein in which one or more crRNA form one or more complexes with the Mmc2 effector protein, and the one or more crRNAs from the complexes hybridize to the one or more target RNA, and ii) one or more engineered or non-naturally occurring Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs, wherein one or more crRNAs is capable of hybridizing with one or more target RNA; and c) detecting the complex bound to the one or more target RNA. Detecting the bound complex is indicative of the presence of one or more target RNA in the sample.
  • methods of diagnosis of an individual suspected of having a disease include: a) providing a sample comprising one or more RNA from the individual; b) contacting the one or more RNA with: i) one or more Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs, wherein one or more crRNAs is capable of hybridizing with one or more RNA from the individual, and ii) a Mmc2 effector protein, or one or more nucleotide sequences encoding a Mmc2 effector protein in which one or more crRNA form one or more complexes with the Mmc2 effector protein, and the one or more crRNAs from the complexes hybridize to the one or more RNA from the individual; and c) detecting the complex bound to the one or more target nucleic acid, in which the binding is indicative of a disease.
  • the Mmc2 effector protein, one or more crRNA or both Mmc2 effector protein and one or more crRNA comprises a detectable label.
  • the detectable label is part of a binding pair, in which binding of the binding pair members is detectable.
  • detecting the bound complex comprises detecting the detectable label.
  • one or more crRNAs and Mmc2 effector protein are differentially labeled.
  • one or more crRNAs are differentially labeled.
  • RNA from the individual or from a pathogenic organism infecting the individual with: a) one or more Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs in which one or more crRNAs is capable of hybridizing with one or more RNA from the individual or one or more RNA of the pathogenic organism, and b) an Mmc2 effector protein, or one or more nucleotide sequences encoding an Mmc2 effector protein in which one or more crRNAs form one or more complexes with the Mmc2 effector protein, and wherein one or more crRNAs from the complexes hybridize to one or more RNA from the individual or one or more RNA of the pathogenic organism.
  • the complexes cause cleavage of one or more RNA from the individual or one or more RNA of the pathogenic organism.
  • the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual is a non-human mammal. In some embodiments, the non-human mammal is a domesticated animal. Non-limiting examples of non-human mammals include monkeys, rats, mice, guinea pigs, rabbits, dogs, cats, cows, pigs, horses, elephants. In some embodiments, the individual is an animal that is not a mammal. Non-limiting examples of non-mammal animals include reptiles, birds, amphibians.
  • the pathogen(s) infecting the individual is one or more types of bacteria.
  • one or more crRNA hybridizes to the RNA of one or more types of bacteria.
  • one or more bacterial RNA hybridized to one or more crRNA is cleaved by the Mmc2 effector.
  • Mmc2 effector upon binding to one or more crRNA and one or more target RNAs from one or more types of bacteria cleaves non-target bacterial RNA that are not complimentary to crRNA.
  • the bacteria are multi-drug resistant.
  • Mmc2 effector upon binding to one or more crRNA and one or more target RNAs from one or more types of bacteria cannot cleave non-target bacterial RNA that are not complimentary to crRNA.
  • contacting of one or more RNA of one or more types of bacteria include infecting one or more types of bacteria with one or more bacteriophages comprising: i) one or more Type VI CRISPR-Cas polynucleotide sequences comprising one or more crRNAs in which one or more crRNAs is capable of hybridizing with one or more bacterial RNA and ii) or one or more nucleotide sequences encoding a Mmc2 effector protein.
  • one or more Type VI CRISPR-Cas polynucleotide sequences comprising one or more crRNAs and/or said nucleotide sequences encoding the Mmc2 effector protein are operably linked to one or more regulatory elements.
  • the regulatory elements are inducible.
  • the treatment comprises inducing the expression of one or more crRNA, nucleic acid encoding said Mmc2 effector protein, or both.
  • inducing the expression of one or more crRNA, nucleic acid encoding said Mmc2 effector protein, or both results in cellular apoptosis.
  • the individual is diagnosed with cancer.
  • the cleavage of cellular RNA bound to target specific crRNA and/or non-specific cleavage of cellular RNA that are not hybridized to crRNA by Mmc2 effector induces apoptosis.
  • the apoptosis is induced to cancer cells.
  • the individual is diagnosed as being infected with RNA virus.
  • RNA virus include Ebola virus, Zika virus, Dengue virus, Measles virus, Mumps virus, Human respiratory syncytial virus, Rabies virus, Polio virus, Hepatitis A virus, Hepatitis C virus, SARS virus, West Nile virus, and Rota virus.
  • the treatment comprises cleaving of viral RNA by Mmc2 effector protein.
  • modified cells are modified or engineered to comprise or express, optionally inducibly or constitutively a composition or a component thereof of the following: a) one or more Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs in which one or more crRNAs is capable of hybridizing with one or more target RNAs, and b) an Mmc2 effector protein, or one or more nucleotide sequences encoding an Mmc2 effector protein in which one or more crRNAs form one or more complexes with the Mmc2 effector protein, and one or more crRNAs hybridize to the one or more target RNAs.
  • the cell comprises a prokaryotic cell.
  • the cell comprises a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is infected with a virus.
  • the cell is infected with an RNA virus.
  • the cell is a cancer cell.
  • the polynucleotides encoding Mmc2 effector and the crRNA are operably linked to regulatory elements.
  • the regulatory elements regulating the expression of the Mmc2 transcript and the transcript comprising crRNA are different.
  • the regulatory elements are inducible.
  • the regulatory elements are inducible by environmental factors.
  • the regulatory elements are regulated by light, temperature, pH, a compound or nutrient present in or absent from the media, or a combination thereof.
  • the regulatory elements are responsive to a compound or nutrient present in or absent from the media.
  • the regulatory elements are regulated by a sugar, an organic acid, a fatty acid, an amino acid, a lipid, a hydrocarbon, phosphate, nitrate, ammonium, nitrogen, sulfur, carbon dioxide, a metal, a quorum-sensing compound, a phenolic compound, a flavonoid, a protein or peptide, or any combination thereof.
  • the cell death is due to apoptosis.
  • the apoptosis results from inducing the expression of Mmc2 effector RNA and the crRNA.
  • one or more crRNA expressed inside the cell can hybridize to one or more abundant RNA inside the cell, e.g., rRNA. In some embodiments, one or more crRNA expressed inside the cell can hybridize to one or more less abundant RNA.
  • the modification of the cells results in (i) cell death (ii) the cell comprising altered transcription or translation of at least one RNA product; (iii) the cell comprising altered transcription or translation of at least one RNA product in which the expression of the at least one product is increased; or (iv) the cell comprising altered transcription or translation of at least one RNA product in which the expression of the at least one product is decreased.
  • the modified cells are cancer cells and one or more crRNA expressed inside the cancer cells hybridize to one or more RNA comprising genetic abnormalities that are causative of cancer.
  • hybridization of one or more crRNA to one or more RNA in the cancer cell causes cleavage of one or more RNA in the cancer cell.
  • the cleavage of one or more RNA in the cancer cell causes apoptotic cell death of the cancer cell.
  • recombinant microorganism genetically engineered for controlled biocontainment in which the microorganism comprises two or more polynucleotides heterologous to the microorganism.
  • One of the two or more heterologous polynucleotides encode Type VI CRISPR-Cas polynucleotide sequences comprising one or more crRNAs, and the other heterologous polynucleotide encodes a Mmc2 effector protein.
  • Two polynucleotides are operably linked to regulatory elements.
  • the regulatory elements are different for each of the two polynucleotides.
  • the regulatory elements are the same for both polynucleotides.
  • the regulatory elements are inducible. In some embodiments, the regulatory elements are inducible by environmental factors. In some embodiments, the regulatory elements are regulated by light, temperature, pH, a compound or nutrient present in or absent from the media, or a combination thereof. In some embodiments, the regulatory elements are responsive to a compound or nutrient present in or absent from the media.
  • the regulatory elements are regulated by a sugar, an organic acid, a fatty acid, an amino acid, a lipid, a hydrocarbon, phosphate, nitrate, ammonium, nitrogen, sulfur, carbon dioxide, a metal, a quorum-sensing compound, a phenolic compound, a flavonoid, a protein or peptide, or any combination thereof.
  • the methods include introducing Mmc2 CRISPR system as described herein to target one or more plant genes 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 known in the art (See Guerineau F., Methods Mol
  • 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
  • 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); flower
  • plants used in phytoremediation e.g., heavy metal accumulating plants
  • oil crops e.g., sunflower, rape seed
  • plants used for experimental purposes e.g., Arabidopsis.
  • the methods and Mmc2 CRISPR systems can be used over a broad range of plants, such as for example with dicotyledonous plants belonging to the orders Magniolales, Miciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violates, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Ju
  • the methods and Mmc2 CRISPR systems can be used with monocotyledonous plants such as those belonging to the orders Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales, or with plants belonging to Gymnospermae, e.g those belonging to the orders Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales.
  • RNA from the microorganism encodes a Type VI CRISPR-Cas polynucleotide sequence comprising one or more crRNAs in which one or more crRNA is capable of hybridizing with one or more RNA from the microorganism and another polynucleotide encodes a Mmc2 effector protein.
  • one or more regulatory elements is regulated by light, temperature, pH, a compound or nutrient present in or absent from the media, or a combination thereof.
  • one or more regulatory element is responsive to a compound or nutrient present in or absent from the media.
  • one or more regulatory element is regulated by a sugar, an organic acid, a fatty acid, an amino acid, a lipid, a hydrocarbon, phosphate, nitrate, ammonium, nitrogen, sulfur, carbon dioxide, a metal, a quorum-sensing compound, a phenolic compound, a flavonoid, a protein or peptide, or any combination thereof.
  • the microorganism is a photosynthetic microorganism.
  • the photosynthetic microorganism is a eukaryotic microalga.
  • the eukaryotic microalga is a species of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Borodinella, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Hymenomonas, Isochrysis,
  • the photosynthetic microorganism is a cyanobacterium.
  • the cyanobacterium is an Acaryochloris, Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeothece, Halospirulina, Iyengariella, Leptolyngbya, Limnothrix
  • the Type VI CRISPR-Cas polypeptide is used to target essential genes of the microorganism.
  • Essential genes can be the ones which when are attenuated negatively affects the growth of the microorganism.
  • Non-limiting examples of essential genes include ribosomal RNA gene, tRNA gene, DNA polymerase gene, RNA polymerase gene.
  • the microorganism is a bacterium.
  • the bacterium is genetically engineered by infecting the bacterium with one or more bacteriophage that delivers one or more Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs which are capable of hybridizing with one or more RNA from the bacterium and one or more nucleotide sequences encoding a Mmc2 effector protein.
  • the biocontainment includes expressing one or more crRNA and the Mmc2 effector protein in the microorganism.
  • the Mmc2 effector forms a complex with one or more crRNA, and one or more crRNA from the complex hybridizes to one or more RNA of the microorganism and the Mmc2 protein cleaves one or more RNA of the microorganism.
  • cleavage of one or more RNA from the microorganism induces apoptosis and contains the propagation of the microorganism.
  • the non-naturally occurring or genetically engineered vector system or the CRISPR-Cas system is used for i) RNA sequence specific interference, (ii) RNA sequence specific gene regulation, (iii) screening of RNA or RNA products or lincRNA or non-coding RNA, or nuclear RNA, or mRNA, (iv) mutagenesis, (v) Fluorescence in situ hybridization, (vi) breeding, (vii) in vitro or in vivo induction of cell dormancy, (viii) in vitro or in vivo induction of cell cycle arrest, (ix) in vitro or in vivo reduction of cell growth and/or cell proliferation, (x) in vitro or in vivo induction of cell anergy, (xi) in vitro or in vivo induction of cell apoptosis, (xii) in vitro or in vivo induction of cell necrosis, (xiii) in vitro or in
  • crRNAs hybridize to target RNA in a sequence-specific manner.
  • the target RNA upon hybridization of crRNAs to target RNA, the target RNA is cleaved by Mmc2 effector protein distal to the hybridization site of the crRNA to the target RNA.
  • RNA other than the target RNA upon hybridization of crRNAs to target RNA, RNA other than the target RNA is cleaved by Mmc2 effector protein.
  • two or more crRNA hybridize to different regions of the same target RNA.
  • two or more crRNA hybridize to different target RNA.
  • the RNA cleaved by Mmc2 effector protein is a single-stranded RNA (ssRNA), partially single-stranded RNA, or RNA with a stem-loop structure.
  • either the target RNA or the non-target RNA cleaved by the Mmc2 effector protein is pre-mRNA, mRNA, tRNA, ribosomal RNA, small nuclear RNA, microRNA (miRNA), small nucleolar RNA (snoRNA), double- stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmic RNA (scRNA), or small interfering RNA (siRNA).
  • the RNA cleaved by Mmc2 effector protein is viral RNA.
  • the viral RNA is present in a cell and heterologous to the cell.
  • the viral RNA is from an RNA virus.
  • Non-limiting examples of RNA virus include Ebola virus, Zika virus, Dengue virus, Measles virus, Mumps virus, Human respiratory syncytial virus, Rabies virus, Polio virus, Hepatitis A virus, Hepatitis C virus, SARS virus, West Nile virus, and Rota virus.
  • the viral RNA is from a DNA virus.
  • DNA virus include Hepatitis B virus, Herpes virus, e.g., HSV-1, HSV-2, Adeno virus, human papilloma virus, varicella-zoster virus, Epstein-Barr virus.
  • the RNA cleaved by Mmc2 effector protein is rRNA. In some embodiments, the RNA cleaved by Mmc2 effector protein is mRNA in a cell. In some embodiments, the cellular mRNA can be a mRNA encoding a protein important for cell viability. In some embodiments, the cellular mRNA can be a mRNA encoding a protein involved in replication, transcription, cell division, protein synthesis, or electron transport.
  • the cellular mRNA encodes DNA polymerase, DNA gyrase, DNA single-strand binding protein, histone, topoisom erase, RNA polymerase, RNA binding protein, ribosomal protein, or cytochrome P450.
  • the Mmc2 effector protein lacks RNA cleavage activity. In some embodiments, the Mmc2 effector protein lacking the RNA cleavage activity can form complex with crRNA. In some embodiments, the Mmc2 effector protein remains bound to the target RNA, but does not cleave the target RNA. In some embodiments, the Mmc2 protein may comprise mutations in the HEPN domains. In some embodiments of the above aspects, the Mmc2 effector protein further comprises RNA modifying enzymes. In some embodiments, the RNA modifying enzyme has site-specific RNA editing activity. In some embodiments, the Mmc2 effector protein can be a fusion protein. In some embodiments, the Mmc2 effector protein can be fused to RNA modifying proteins. Non-limiting examples of RNA modifying protein includes RNase, RNA binding protein, RNaseH.
  • one or more Mmc2 CRISPR RNA is linked to direct repeat sequences. In some embodiments, one or more Mmc2 CRISPR RNA is linked to multiple repeat sequences. In some embodiments, the Mmc2 CRISPR RNA with one or more direct repeat sequences are processed by endonucleolytic cleavage to produce mature crRNA.
  • one or more target RNA is inside a cell.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a cancerous cell.
  • modification of target RNA by Mmc2 effector protein inside a cell causes cell death.
  • the vector system or the Mmc2 CRISPR-Cas system comprises multiple crRNA that can hybridize to multiple different target RNA.
  • the Mmc2 effector of the vector system or the Mmc2 CRISPR-Cas system can cleave multiple target RNA to which one or more crRNA hybridizes.
  • the vector system or the Mmc2 CRISPR-Cas system comprises multiple crRNA that can hybridize to multiple sites of the same target RNA and the Mmc2 effector cleaves at multiple sites of the same target RNA.
  • the Mmc2 effectors cleave at multiple sites distal to the hybridization site of the crRNA to the target RNA.
  • one or more Type VI CRISPR-Cas polynucleotide sequences comprising or encoding one or more crRNAs and/or said nucleotide sequences encoding said Mmc2 effector protein is operably linked to one or more regulatory element.
  • regulatory elements include a promoter, an enhancer, an internal ribosomal entry sites (IRES), a 5 '-untranslated region, and a 3 '-untranslated region.
  • the regulatory element is inducible.
  • one or more nucleotide sequences encoding the Mmc2 effector protein is codon optimized for expression in a eukaryotic cell.
  • Mmc2 effector protein comprises one or more nuclear localization signal(s) (NLS(s)).
  • non-naturally occurring or engineered system is delivered inside a cell or a cellular organelle by electroporation, nucleofection, lipofection, calcium phosphate precipitation, or via a delivery vehicle comprising liposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun or one or more viral vector(s).
  • FIG. 1 Schematic of Mmc2 system architectures and gene regions. Representative contigs are shown indicating the Mmc2 effector, CRISPR array, Cas genes and flanking gene CDS.
  • FIG. 1 Phylogenetic analysis of Mmc2 relative to all known Class II CRISPR systems. Maximum likelihood phylogenetic analysis was performed using a MAFFT alignment of Class II CRISPR effector protein sequences. Mmc2 formed a mono- phyletic group distinct from all known Class II effector proteins.
  • Figure 3 Phylogenetic analysis of Mmc2 relative to all known Type VI CRISPR systems. Maximum likelihood phylogenetic analysis was performed using a MAFFT alignment of Type VI CRISPR effector protein sequences. Bootstrap analysis was performed with 100 pseudoreplicates and gives a statistical measure of support for the tree topology. Mmc2 formed a mono-phyletic group distinct from all known Type VI CRISPR effector proteins that had high statistical support.
  • Figure 4 Unrooted phylogenetic analysis of Mmc2 relative to all known Type VI CRISPR systems. Maximum likelihood phylogenetic analysis was performed using a MAFFT alignment of Type VI CRISPR effector protein sequences. Bootstrap analysis was performed with 100 pseudoreplicates and gives a statistical measure of support for the tree topology. Mmc2 formed a mono-phyletic group distinct from all known Type VI CRISPR effector proteins that had high statistical support.
  • Figure 5 Phylogenetic analysis of Mmc2 relative to all known Type VI CRISPR systems using an un-gapped alignment. Positions with any gap in the alignment were masked to generate a 215 bp un-gapped alignment across all Type VI CRISPR Effectors. Maximum likelihood analysis of this alignment produced a tree with topology consistent with the gapped- non-normalized alignment demonstrating that phylogenetic separation of Mmc2 from other Type VI CRISPR effectors is not due to the smaller size of Mmc2 proteins in general.
  • Mmc2 Effectors form a closed network, relative to all other known Type VI CRISPR effectors ( Figure 6, top panel).
  • the Mmc2 network was very robust, since an alignment threshold of 343 was needed to generate an edge between Mmc2 and another Type VI
  • FIG. 7 RNAseq data for E. siraeum (DSM15702) Mmc2 CRISPR region. Zoom region shows predicted processed form of the mature crRNA containing 30 nucleotides of the 5' direct repeat followed by 20-25 nt of the 3' spacer.
  • FIG. 8 RNAseq analysis of E. coli expressing a synthetic CRISPR array in the presence of dEs5Mmc2. Mapped reads show predicted processed form of the mature crRNA containing 30 nt of the 5' direct repeat followed by 20-25 nt of the 3' spacer. Two processed crRNA' s were produced from a single RNA transcript - one derived from each of the direct repeats.
  • FIGS 9A-9B A) Alignment of Mmc2 CRISPR repeat sequences. Shading represents predicted stem region of a hairpin structure. The white text represents predicted unpaired bases within the stem structure. Consensus sequence is represented as a seqLogo. B) Alignment of known Type VI CRISPR repeat sequences. Shading represents predicted stem region of the hairpin structure. White text represents predicted unpaired bases within the stem structure. Consensus sequence is represented as a seqLogo.
  • FIG. 10 Predicted secondary structure of Type VI CRISPR direct repeat sequences. Examples are shown for experimentally validated Mmc2 systems. Arrow indicates determined processing site. Representative examples are given for C2c2 (Casl3a), Casl3b and Casl3c systems.
  • FIG. 11 Schematic of PFS requirements for Type VI CRISPR systems.
  • Members of the C2c2 (Casl3a) sub-type vary with respect to their requirement for a PFS.
  • C2c2 systems with a PFS requirement have a PFS 3' of the target site.
  • Mmc2 systems do not have a strong requirement for specific sequence motifs (PFS) proximal to the target site. This does not exclude the possibility that some Mmc2 systems will have a PFS requirement as is the case for C2c2.
  • Casl3b has a strong PFS requirement both for the 5' and 3' sequences flanking the target sequence. No information is available for the PFS requirements of Casl3c.
  • FIG. 12 Sequence alignment of conserved HEPN domains in Type VI CRIPSR systems. conserved catalytic residues are shown by white letter on blue background and conserved residues with polar side chains (N, Q, H) are highlighted in orange. Amino acid residues with hydrophobic and small side chains (G, S, T, C, A, V, A, V, I, L, M, F, Y, W) are highlighted in yellow; negatively charged side chains (D, E) are highlighted in green.
  • Figure 13 Representation of the location of HEPN domains in various TYPE VI CRISPR Effector proteins. Distribution of HEPN1 and HEPN2 domains across the effector sequences are unique compared to C2c2, Casl3b and Casl3c. Scaling for each subtype was derived from the average lengths taken from the representative sequences shown on Figure 13.
  • FIG. 14 Multiple sequence alignment of C2c2s and Mmc2s. HEPN domains are highlighted in gray boxes. The C2c2 recognition lobe (REC) responsible for crRNA interaction is marked with a bracket. The corresponding region in Mmc2 is poorly conserved and includes numerous large gaps in the alignment relative to C2c2. .
  • REC C2c2 recognition lobe
  • FIG. 15 Schematic of typical in vivo RNA interference assay.
  • E. coli cells are co-transformed with a plasmid expressing an effector (e.g. Mmc2) and a plasmid encoding a minimal synthetic crRNA cassette.
  • Complex formation between the effector and a compatible crRNA directs cleavage of the RNA target based on hybridization with the crRNA.
  • the RNA derives from an essential gene, depletion of the RNA results in cell death. Therefore, the relative reduction in transformation frequency indicates activity of the system for RNA-guided RNA interference.
  • RNA-guided RNA-interference of EupMmc2, Es4Mmc2 and Es5Mmc2 in E. coli Relative reduction in transformation frequency indicates activity of system for RNA-guided RNA interference.
  • On-target crRNA encode spacer sequences complementary to ArgS and AspS transcripts, which are essential in E. coli.
  • the random crRNA encodes a spacer sequence that is not complementary to any known transcript in E. coli.
  • the non-cognate crRNA utilizes a direct repeat sequence that is derived from an unrelated CRISPR system. Reductions in transformation frequency were observed for all three Mmc2 systems for both targets. Activity was dependent on using target-specific spacer sequences (on-target) and cognate CRISPR repeat sequences.
  • FIG. 17 Purification of Mmc2 proteins. Mmc2s were affinity purified using a strep tag, followed by cleavage of the tag using SUMO protease. Further size exclusion chromatography (SEC)was performed to remove non-specific RNAse activity.
  • SEC size exclusion chromatography
  • FIG. 18 In vitro demonstration of RNA-guided and RNA interference of Mmc2s.
  • Mmc2 cannot recognize dsDNA as substrate for RNA-guided cleavage or to trigger non-specific RNase activity (collateral effect).
  • Es4Mmc2 and Es5Mmc2 showed no detectable cleavage of the target dsDNA (EtBR filter) when incubated with on-target crRNA complementary to dsDNA (+) strand for 2 hours. Additionally, neither system showed cleavage of the non-target labeled ssRNA (Cyanine filter) reinforcing the absence of an on-target interaction with the dsDNA target.
  • FIG 20 In vitro cleavage of CRISPR array catalyzed by Es4Mmc2.
  • Mmc2 exhibits non-specific RNase activity subsequent to on-target interaction between crRNA and target ssRNA.
  • Etbr filter visualizes both target and non-target ssRNA's, whereas, the cyanine filter visualizes only the labelled non-target ssRNA.
  • FIG. 22 Comparison of RNA-guided RNA-interference activity of Es4Mmc2, dEs4Mmc2, Es5Mmc2 and dEs5Mmc2 in E. coR. Relative reduction in transformation frequency indicates activity of system for RNA-guided RNA interference.
  • On-target crRNA encode a spacer sequence complementary to ArgS transcripts in E. coli.
  • Random crRNA encodes a spacer sequence non-complementary to any transcript in E. coli. N.D. indicates that no viable colonies were detected over the range of dilutions analyzed.
  • Mmc2 CRISPR systems comprising Mmc2 effector proteins that are evolutionarily distinct from all known Type VI effector proteins (e.g., C2c2(Casl3a), Casl3b, Casl3c), the methods and kits using novel Mmc2 CRISPR system.
  • C2c2(Casl3a), Casl3b, Casl3c) the methods and kits using novel Mmc2 CRISPR system.
  • Mmc2 CRISPR systems are represented by several different system architectures.
  • the minimal system is composed of an effector (Mmc2) and a CRISPR array.
  • systems encode Casl, Cas2 genes.
  • the Mmc2 CRISPR systems do not encode Casl, Cas2 genes. ( Figure 1).
  • Mmc2 effectors comprise an RNA-guided RNA- cleaving enzymatic activity.
  • Mmc2 effectors formed a distinct mono- phyletic clade within all known Class II CRISPR systems ( Figure 2).
  • Mmc2 is most related to Type VI CRISPR systems as demonstrated by the genetic distance of the Mmc2 clade relative to C2c2, Casl3b and Casl3c as compared to Type II (Cas9) and Type V (Cpfl, C2cl, C2c3, CasX, CasY) clades.
  • Mmc2 effectors are evolutionarily distinct from all other known Type VI CRISPR systems (Figs. 3-6).
  • the Mmc2 class of effectors have a sequence identity ranging from 17%-98% within the class. Mmc2 effectors have extremely low sequence identities of 4%-9% with other Type VI class of effectors, e.g., C2c2, Casl3b, Casl3c effectors.
  • the average length of Mmc2 Effector proteins is substantially shorter than effector proteins of other Type VI CRISPR systems. In some embodiments, the lengths of the Mmc2 effector proteins are less than 1000 amino acids.
  • Mmc2 effector comprises two conserved RxxxxH motifs that are associated with RNase activity.
  • RxxxxH motifs are characteristic of HEPN (Higher eukaryotes and prokaryotes nucleotide-binding) domains, which are also present in other Type VI CRISPR Effectors (Casl3a, Casl3b, Casl3c) ( Figure 12).
  • the spacing between HEPN catalytic motifs are noticeably different for Mmc2 effectors as compared to other Type VI CRISPR effectors and consistent with the designation of Mmc2 as a distinct subtype ( Figure 13).
  • C2c2 (Casl 3a) which are the only Type VI systems with available crystal structures, showed poor alignment of the N-terminal region ( Figure 14). Interestingly, the N-terminal region of C2c2 effectors contain the domains responsible for crRNA recognition (REC) suggesting that Mmc2 has a distinct structural fold for its REC lobe. Further support can be inferred by the differences in the crRNA sequences between Mmc2 and C2c2 ( Figures 9A & B).
  • Mmc2 effector proteins process the CRISPR array to generate mature crRNA.
  • Mmc2 effector proteins have two RNase motifs: a sequence non-specific RNase activity catalyzed by HEPN domain; and a crRNA processing and RNA binding motifs independent of the HEPN domain.
  • Mmc2 effectors cleave RNA non-specifically once activated by the interaction of the effectors, with crRNA and with target ssRNA.
  • the Mmc2 effectors do not require Protospacer
  • Flanking Sequence a sequence motif proximal to the target site in the ssRNA.
  • the Mmc2 effectors are from Eubacterium sp.
  • Ruminococcus sp. Eubacterium siraeum, Ruminococcus flavefaciens, or Ruminococcus bicirculans.
  • Non-limiting exemplary amino acid sequences of the Type VI Mmc2 sub class of effectors are provided as SEQ ID NOs 1-37.
  • the Mmc2 effector protein comprises an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of naturally occurring Mmc2 effectors. In some embodiments, the Mmc2 effector protein comprises an amino acid sequence that is at least about 60%>, 65%>, 70%), 75%), 80%), 85%o, 90%, or 95% identical to the amino acid sequence of any one of SEQ ID NOs: 1-37.
  • the Mmc2 effector protein comprises an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to at least about 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acids of any one of SEQ ID NOs: 1-37. In some embodiments, the Mmc2 effector protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-37. In some embodiments, the Mmc2 effector protein consists essentially of the amino acid sequence of any one of SEQ ID NOs: 1-37. In some embodiments, the Mmc2 effector protein consists of the amino acid sequence of any one of SEQ ID NOs: 1-37. [0088] Mmc2 CRISPR Arrays
  • Mmc2 CRISPR systems encode a CRISPR array proximal to the Mmc2 effector proteins.
  • the Mmc2 CRISPR array had a repeat sequence of 36 bp.
  • the Mmc2 CRISPR array had a CRISPR repeat sequence of 33 bp in length.
  • C2c2 repeats which span a range of lengths from 31 to 39 bp ( Figure 9B).
  • Casl3b repeats are typically of length 36 bp ( Figure 9B).
  • Casl3c repeat sequences are ⁇ 30bp, which is substantially shorter than other Type VI systems.
  • 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 cytoplasmic RNA (scRNA).
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre- mRNA, and rRNA.
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some more preferred embodiments, the target sequence may be a sequence within a mRNA molecule or a pre-mRNA molecule.
  • a crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence.
  • the 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.
  • the direct repeat sequence may be located downstream (i.e., 3') from the guide sequence or spacer sequence.
  • the mature crRNA is derived from a CRISPR array comprising one or more direct repeats.
  • the CRISPR array is processed from the 5'- or from 3'-end to generate one or more mature crRNA.
  • 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 Mmc2 crRNA is from 15 to 40 nucleotides. In certain embodiments, the spacer length of the crRNA is at least 15, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more nucleotides.
  • the spacer length is from 17 to 20 nucleotides, e.g., 17, 18, 19, or 20 nucleotides, from 20 to 24 nucleotides, e.g., 20, 21, 22, 23, or 24 nucleotides, from 23 to 25 nucleotides, e.g., 23, 24, or 25 nucleotides, from 24 to 27 nucleotides, e.g., 24, 25, 26, or 27 nucleotides, from 27-30 nucleotides, e.g., 27, 28, 29, or 30 nucleotides, from 30-35 nucleotides, e.g., 30, 31, 32, 33, 34, or 35 nucleotides, or longer.
  • the length of the direct repeat sequence of Mmc2 crRNA is from 15 to 40 nucleotides. In certain embodiments, the length of the direct repeat sequence of Mmc2 crRNA is 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In one embodiment, the length of the direct repeat sequence of Mmc2 crRNA is 30, 31, 32, 33, 34, 35, or 36 nucleotides.
  • the crRNA may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
  • a crRNA acid may be modified such as by conjugation, with a labeling component.
  • the crRNA are non-naturally occurring or
  • the crRNA or the polynucleotide encoding or comprising one or more crRNA are heterologous to the cell in which the crRNA or the polynucleotide encoding or comprising one or more crRNA is introduced.
  • the term "collateral effect" in the context of Mmc2 effectors means that in some embodiments, the Mmc2 effector upon binding to one or more crRNA and one or more target RNAs, cleaves one or more non-target RNA that are not complimentary to crRNA.
  • Mmc2 effector protein demonstrates collateral effect.
  • Mmc2 effectors do not demonstrate collateral effect.
  • the Mmc2 effector does not have the ability to cleave non- target RNA when the Mmc2 effector is bound to a crRNA which is hybridized to target RNA.
  • codon optimized sequence is protein sequences e.g., Mmc2 effector proteins, optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal.
  • 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.
  • Examples of codon optimized CRISPR enzymes include SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). 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 RNA-modif ing Mmc2 protein is codon optimized for expression in 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.
  • Various species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for 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 in a sequence encoding a DNA/RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid.
  • a vector encodes a RNA modifying effector protein such as the Type VI RNA-targeting effector protein, in particular Mmc2, or an ortholog or homolog thereof comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • NLSs nuclear localization sequences
  • the RNA-modifying effector protein comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus).
  • each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- 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; the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP ; the hRNPAl M9 NLS having the sequence NQS SNFGPMKGGNFGGRS SGP YGGGGQYF AKPRNQGGY; the sequence
  • the one or more NLSs are of sufficient strength to drive accumulation of the RNA-modifying Mmc2 effector protein in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of LSs in the RNA modifying effector protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the RNA modifying effector protein, 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).
  • 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 RNA modifying complex formation (e.g., assay for RNA cleavage, or assay for altered gene expression activity affected by RNA-modif ing complex formation and/or RNA-modifying Mmc2 effector protein activity), as compared to a control not exposed to the RNA modifying Mmc2 effector protein or RNA modifying complex, or exposed to a RNA modifying effector protein lacking the one or more NLSs.
  • an assay for the effect of RNA modifying complex formation e.g., assay for RNA cleavage, or assay for altered gene expression activity affected by RNA-modif ing complex formation and/or RNA-modifying Mmc2 effector protein activity
  • Mmc2 effector protein complexes and systems are codon optimized.
  • the codon-optimized Mmc2 effector protein further comprises NLS.
  • the codon-optimized Mmc2 effector proteins comprise an NLS attached to the C-terminal of the protein.
  • the codon-optimized Mmc2 effector proteins comprise an NLS attached to the N-terminal of the protein.
  • one or more vectors driving expression of one or more elements of an RNA modifying system are introduced into a host cell such that expression of the elements of the RNA modifying system direct formation of an RNA modifying complex at one or more target sites.
  • an RNA modifying effector enzyme and a crRNA could each be operably linked to separate regulatory elements on separate vectors.
  • RNA(s) of the RNA modifying system can be delivered to a transgenic RNA modifying effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses RNA modifying protein; or an animal or mammal that is otherwise expressing RNA modifying effector protein or has cells containing RNA modifying effector protein, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo RNA modifying effector protein.
  • a transgenic RNA modifying effector protein animal or mammal e.g., an animal or mammal that constitutively or inducibly or conditionally expresses RNA modifying protein; or an animal or mammal that is otherwise expressing RNA modifying effector protein or has cells containing RNA modifying effector protein, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo RNA modifying effector protein.
  • RNA modifying system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a RNA modifying effector protein and the crRNA, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).
  • the RNA modifying effector protein and the guide RNA may be operably linked to and expressed from the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a RNA modifying system are as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667).
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • insertion sites such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • one or more insertion sites e.g., about or more than about 1, 2, 3, 4, 5,
  • a vector comprises two or more insertion sites, to allow insertion of a guide sequence at each site.
  • the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target RNA modifying activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6,
  • guide sequences 7, 8, 9, 10, 15, 20, or more guide sequences.
  • about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a RNA modifying effector protein, or crRNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a particle or nanoparticle complex, RNA modifying effector protein mRNA can be delivered prior to the crRNA to give time for RNA modifying effector protein to be expressed, RNA modifying effector protein mRNA might be administered 1-12 hours
  • RNA modifying effector protein mRNA and crRNA can be administered together.
  • a second booster dose of crRNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration of RNA modifying effector protein mRNA + guide RNA. Additional administrations of RNA modifying effector protein mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome and/or transcriptome modification.
  • Mmc2 effector refers to an RNA-guided RNA-targeting polypeptide possessing enzymatic activity.
  • enzymatic activity include RNA binding, endonuclease, nickase, integrase, or transposase activity.
  • CRISPR array refers to the DNA segment which includes all of the CRISPR repeats and spacers, starting with the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer sequence in a CRISPR array is located between two repeats.
  • CRISPR repeat As used herein, the terms “CRISPR repeat,” “direct repeat,” “repeat sequence,” or “repeat” have the conventional meaning as used in the art, i.e., multiple short direct repeating sequences, which show very little or no sequence variation within a given CRISPR array.
  • CRISPR spacer As used herein, “CRISPR spacer,” “spacer sequence,” or “spacer” refer to the non- repetitive sequences that are located between the repeats of a CRISPR array.
  • the term "crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components" of a Type VI CRISPR-Cas locus Mmc2 effector protein comprises any polyribonucleotide sequence having sufficient complementarity with a target RNA 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 includes 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.
  • any suitable algorithm for aligning sequences non-limiting example of which includes 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.
  • target RNA means RNA that is capable of hybridizing to one or more crRNA.
  • the target RNA is modified by the Mmc2 CRISPR system.
  • the target RNA is endonucleolytically cleaved by one or more Mmc2 effectors.
  • hybridize or “hybridization” refers to the pairing of substantially complementary nucleotide sequences (strands of nucleic acid) to form a duplex or heteroduplex through the formation of hydrogen bonds between complementary base pairs.
  • Hybridization and the strength of hybridization is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T.sub.m of the formed hybrid.
  • An oligonucleotide or polynucleotide e.g., a probe or a primer
  • Hybridize to the target nucleic acid under suitable conditions. See e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Press, Plainview, N.Y.
  • modifying means binding of a Mmc2 protein to that RNA.
  • modifying a RNA further included endonucleolytic cleavage of the RNA.
  • the terms “about” or “approximately” when referring to any numerical value are intended to mean a value of plus or minus 10% of the stated value.
  • “about 50 degrees C” encompasses a range of temperatures from 45 degrees C to 55 degrees C, inclusive.
  • “about 100 mM” encompasses a range of concentrations from 90 mM to 110 mM, inclusive. All ranges provided within the application are inclusive of the values of the upper and lower ends of the range.
  • an "isolated" biomolecule such as an isolated protein or nucleic acid, is a biomolecule removed from the context in which the biomolecule exists in nature.
  • an isolated protein or nucleic acid molecule is removed from the cell or organism with which it is associated in its natural state.
  • An isolated biomolecule can be, in some instances, partially or substantially purified, for example, an isolated nucleic acid molecule can be a nucleic acid sequence that has been excised from the chromosome, genome, or episome that it is integrated into in nature.
  • a recombinant or "engineered” nucleic acid molecule is a nucleic acid molecule that has been altered through human intervention.
  • a recombinant nucleic acid molecule 1) includes conjoined nucleotide sequences that are not conjoined in nature, 2) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence, or 3) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence.
  • a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
  • a recombinant or "engineered” organism is an organism into which one or more recombinant or “engineered” nucleic acid molecules has been introduced.
  • a "homolog" of a gene or protein refers to its functional equivalent in another species.
  • a "variant" of a gene or nucleic acid sequence is a sequence having at least 65% identity with the referenced gene or nucleic acid sequence, and can include one or more base deletions, additions, or substitutions with respect to the referenced sequence. Variants also include chimeric genes that include sequences from two or more sources. Variants also include codon-optimized genes, and genes containing mutations, insertions, deletions, or substitutions, either naturally-occurring or recombinant. A variant can be a naturally-occurring variant or the result of a spontaneous or induced mutation.
  • Induced mutations can be created using methods known in the art for mutagenesis of organisms or cells (for example, using gamma or UV irradiation or chemical mutagens such as 5-bromo deoxyuridine, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES), nitrosoguanidine (NTG), ICR compounds, etc., or can be introduced using genetic engineering techniques, such as gene synthesis, in vivo single-strand repair techniques, polymerase-based amplification at error- permissive temperature and/or polymerase-based amplification using primers that incorporate base changes.
  • EMS ethyl methane sulfonate
  • MMS methyl methane sulfonate
  • DES diethylsulfate
  • NGT nitrosoguanidine
  • ICR compounds etc.
  • genetic engineering techniques such as gene synthesis, in vivo
  • a "variant" of a peptide or protein is a peptide or protein sequence that varies at one or more amino acid positions with respect to the reference peptide or protein.
  • a variant can be a naturally-occurring variant or can be the result of spontaneous, induced, or genetically engineered mutation(s) to the nucleic acid molecule encoding the variant peptide or protein.
  • a variant peptide can also be a chemically synthesized variant.
  • the degree of amino acid or nucleic acid sequence identity can be determined by various computer programs for aligning the sequences to be compared based on designated program parameters. For example, sequences can be aligned and compared using the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), or the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the default parameters of the BLAST programs can be used.
  • the BLASTP defaults are: word length (W), 3; expectation (E), 10; and the BLOSUM62 scoring matrix.
  • the TBLASTN program uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix, (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &
  • the smallest sum probability provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) The smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, preferably less than about 0.01, and more preferably less than about 0.001.
  • Exogenous in the context of a gene or protein is a gene or protein that is not derived from the host organism species.
  • a "heterologous" gene or nucleic acid sequence is a gene or sequence from a different source than the host organism it is introduced into, or from a different source than another nucleic acid sequence with which is juxtaposed in a nucleic acid construct.
  • a gene of one species introduced into another species may be referred to as a heterologous gene.
  • a nucleic acid molecule that includes a gene operably linked to a promoter that is not the natural promoter for the gene (not the promoter linked to the gene in its natural state) is also referred to herein as a heterologous nucleic acid molecule or sequence, even though the gene may be derived from the same species as the host organism.
  • a gene that is "codon-optimized" for expression in an organism is a gene whose nucleotide sequence has been altered with respect to the original nucleotide sequence, such that one or more codons of the nucleotide sequence has been changed to a different codon that encodes the same amino acid, in which the new codon is used more frequently in genes of the organism of interest than the original codon.
  • the degeneracy of the genetic code provides that all amino acids except form methionine and tryptophan are encoded by more than one codon.
  • arginine, leucine, and serine are encoded by different six different codons; glycine, alanine, valine, threonine, and proline are encoded by four different codons.
  • Many organisms use certain codons to encode a particular amino acid more frequently than others. Without limiting any aspects of the invention to any particular mechanism, it is believed that some tRNAs for a given amino acid are more prevalent than others within a particular organism, and genes requiring a rare tRNA for translation of the encoded protein may be expressed at a low level due in part to a limiting amount of the rare tRNA.
  • a gene may be "codon-optimized” to change one or more codons to new codons ("preferred codons") that are among those used more frequently in the genes of the host organism (referred to as the "codon preference" of the organism).
  • a "codon-optimized” gene or nucleic acid molecule of the invention need not have every codon altered to conform to the codon preference of the intended host organism, nor is it required that altered codons of a "codon-optimized” gene or nucleic acid molecule be changed to the most prevalent codon used by the organism of interest.
  • a codon-optimized gene may have one or more codons changed to codons that are used more frequently that the original codon(s), whether or not they are used most frequently in the organism to encode a particular amino acid.
  • a "regulatory element” as used herein includes 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).
  • 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. Such regulatory element would be operably configured to express e.g., a Mmc2 effector polypeptide, or nucleic acid component(s). For example, and without limitation, expression of a Mmc2 effector polypeptide in a human cell is accomplished using CMV expression vectors.
  • a U6 promoter is fused to a crRNA sequence.
  • Other common regulatory elements useful in vectors having the adaptor module, CRISPR array or effector module include: pol I, II or pol III promoters such as U6 and HI, RSV LTR promoter and/or enhancer), CMV promoter/enhancer, SV40 promoter, ⁇ -actin promoter, DHFR promoter, PGK promoter, and the EFla promoter, R-U5' segment in LTR of HTLV-I, SV40 enhancer, human beta actin, chicken beta actin, CAG, Ubc, TRE, UAS, Polyhedrin, CaMKIIa, GAL1, TEF, Ac5, GDS, ADHl, CaMV35S, Ubi, and others generally known to those of skill in the art.
  • the regulatory elements are inducible. In some embodiments, the regulatory elements are inducible by environmental factors. In some embodiments, the regulatory elements are regulated by light, temperature, pH, a compound or nutrient present in or absent from the media, or a combination thereof. In some embodiments, the regulatory elements are responsive to a compound or nutrient present in or absent from the media.
  • the regulatory elements are regulated by a sugar, an organic acid, a fatty acid, an amino acid, a lipid, a hydrocarbon, phosphate, nitrate, ammonium, nitrogen, sulfur, carbon dioxide, a metal, a quorum-sensing compound, a phenolic compound, a flavonoid, a protein or peptide, or any combination thereof.
  • Inducible promoters are described in U.S. Patent No. 8,975,061, which is incorporated by reference in its entirety.
  • Vector refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide capable of being delivered to a target cell, either in vitro, in vivo or ex -vivo.
  • the heterologous polynucleotide can comprise a sequence of interest and can be operably linked to another nucleic acid sequence such as promoter or enhancer and may control the transcription of the nucleic acid sequence of interest.
  • a vector need not be capable of replication in the ultimate target cell or subject.
  • the term vector may include expression vector and cloning vector.
  • Suitable expression vectors are well-known in the art, and include vectors capable of expressing a polynucleotide operatively linked to a regulatory sequence, such as a promoter region that can regulate expression of such DNA.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA.
  • Appropriate expression vectors include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • Promoter refers to a segment of DNA that controls transcription of polynucleotide to which it is operatively linked. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary eukaryotic promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter.
  • CMV cytomegalovirus
  • MMTV mouse mammary tumor virus
  • MMLV Moloney murine leukemia virus
  • Exemplary promoters suitable for use with prokaryotic hosts include T7 promoter, beta-lactamase promoter, lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • Detectable label refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest. In some cases, the detectable label may be detected directly. In other cases, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label. Detectable labels may be isotopes, fluorescent moieties, colored substances, and the like.
  • means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.
  • pathogenic organism means organisms that can cause disease in an individual.
  • pathogenic organisms include bacteria, virus, protozoa.
  • Non-limiting exemplary bacteria pathogenic to humans is listed in Table 1 below.
  • NGU Nongonococcal urethritis
  • EHEC E.g., EHEC
  • Tularemia Fever, ulceration at entry site
  • Klebsiella pneumoniae • Klebsiella pneumonia, with significant lung necrosis
  • implanted prostheses e. epidermidis
  • Neonatal meningitis Neonatal sepsis
  • Yersinia pest is • Bubonic plague
  • a Casl HMM was trained on a combination of proprietary and public protein sequence data (about 75.8 million proteins).
  • HMMER v.3.1b2 was used to iteratively search the dataset for Casl, updating the HMM each time. This behavior recapitulates the steps taken by Jackhmmer (https://www.ebi.ac.uk/Tools/hmmer/search/jackhmmer). The loop runs five times or until the model converges, whichever comes first. The output of this search contained contigs very likely to contain Casl . These contigs were run through PILER-CR v.1.06 to identify the subset likely to have CRISPR repeat sequences.
  • HMM hidden Markov model
  • Mmc2 protein sequences were aligned with reference sequences for all known Class II CRISPR systems, Type II - Cas9; Type V - Cpfl(Casl2a), C2cl(Casl2b), CasY, CasX, C2c3(Casl2c), Type VI - C2c2/Casl3a, Casl3b, Casl3c.
  • Cpfl sequences were taken from Zetsche et al. (2015) Cell, 163(3), 759-771.
  • C2cl and C2c3 were taken from Shmakov et al. (2015) Molecular Cell, 60(3), 385-397.
  • CasX and CasY sequences were taken from Burstein et al. (2017) Nature, 1-20.
  • C2c2 sequences were taken from Shmakov et al. (2015) Molecular Cell, 60(3), 385-397 & East-Seletsky et al. (2017) Molecular Cell, 66(3), 373-383.e3.
  • Casl3b sequences were taken from Smargon et al. (2017) Molecular Cell, 1-21.
  • Casl3c sequences were taken from Shmakov et al. (2017) Nature Publishing Group, 1-14.
  • Mmc2 effectors are most related to Type VI CRISPR systems as demonstrated by the genetic distance of the Mmc2 clade relative to C2c2, Casl3b and Casl3c as compared to Type II (Cas9) and Type V (Cpfl, C2cl, C2c3, CasX, CasY) clades.
  • Mmc2 effectors form a closed network, relative to all other known Type VI CRISPR effectors ( Figure 6, top panel).
  • the Mmc2 network was very robust, since an alignment threshold of 343 was needed to generate an edge between Mmc2 and another Type VI CRISPR effector ( Figure 6, bottom panel).
  • an alignment threshold of 343 was needed to generate an edge between Mmc2 and another Type VI CRISPR effector ( Figure 6, bottom panel).
  • C2c2, Casl3b and Casl3c have collapsed into a single network.
  • this analysis supports the claim that Mmc2 is a distinct effector and a new subtype of Type VI CRISPR systems.
  • Mmc2 effectors have sequence identities ranged from 17 % to 98% within the class.
  • Mmc2 effectors as a class is very distinct from known Type VI systems, with extremely low sequence identities of 4% to 9% when aligned to C2c2, Casl3b and Casl3c Effectors.
  • Each Mmc2 effector protein sequence identified from the proprietary database was used as a query against the NCBI non-redundant database using the Blastp algorithm with default settings.
  • the analysis identified 7 full length Mmc2 effectors with E value ⁇ 0.01 and shown in Table 3.
  • Three additional partial systems referred to as Eup2Mmc2 (accession: CDF 15656), Rsp3Mmc2 (accession: SCH71549) and Rsp4Mmc2 (accession: SCJ16097) were also identified.
  • Eup2Mmc2 accession: CDF 15656
  • Rsp3Mmc2 accession: SCH71549
  • Rsp4Mmc2 accession: SCJ16097
  • Mmc2 effector proteins are ⁇ 1000 aa long and is a hallmark of Mmc2 systems within Type VI CRISPR systems (Table 2 & 3).
  • CRISPR arrays were detected using the bioinformatics software CRISPRfinder (http://crispr.i2bc.paris-saclay.fr/Server/ and CRISPRdetect
  • C2c2 repeats span a range of lengths from 31 to 39 bp ( Figure 9B). Similar to Mmc2, Casl3b repeats are typically of length 36 bp ( Figure 9B). Casl3c repeat sequences are ⁇ 30bp, which is substantially shorter than other Type VI systems.
  • RNA predictions using the Vienna RNA suite http://rna.tbi.univie.ac.at/) plug-in within Gensious RIO software (www.Geneious.com) provided support for a conserved hairpin structure in the middle of the CRISPR RNA repeat sequence (Fig 9A).
  • Predicted hairpin structures differed from those of other Type VI CRISPR system crRNA's. Specifically, the stem region of Mmc2 crRNA hairpins typically span 7-9 bp, which is substantially larger than the 4-5 bp typically predicted for C2c2 crRNA ( Figure 9B).
  • the mature Mmc2 crRNA's is composed of an -30 bp handle derived from the CRISPR repeat sequence that is 5' of a -22-25 bp spacer region. This orientation of processed crRNA is similar to C2c2, but differs markedly from Casl3b which utilizes crRNA with the spacer sequence 5' of the CRISPR repeat sequence ( Figure 11). No data is available for crRNA processing of casl3c systems.
  • Mmc2 systems were assessed for the presence of tracrRNA by analyzing intergenic regions for sequence with partial complementarity to CRISPR repeat sequences. Suboptimal alignments failed to uncover strong evidence for an anti-repeat sequence typical of tracrRNA from other systems (i.e. Cas9, C2cl). Based on this analysis, Mmc2 systems were considered unlikely to require accessory RNAs for functional interference activity.
  • HEPN Higher eukaryotes and prokaryotes nucleotide-binding domains, which are also present in other Type VI CRISPR effectors (Casl3a, Casl3b, Casl3c) ( Figure 12).
  • the HEPN superfamily includes extremely diverse sequences with highly conserved catalytic motifs shown to possess RNase activity.
  • Mmc2 is distinguished from other Type VI CRISPR Effectors by showing conservation for a Q immediately after the R in the HEPN1 domain in 75% of the sequences.
  • Engineered Mmc2 systems were tested in E. coli for their ability to exhibit RNA-guided cleavage of an RNA transcript derived from a chromosomally encoded essential gene (Figure 15).
  • the test system was composed of two parts: 1) synthesized Effector cloned into a low copy vector under control of an inducible PTet promoter and 2) a synthetic processed CRISPR RNA encoding a non-natural spacer was expressed from a constitutive promoter on a med copy vector.
  • the Mmc2 effector proteins, crRNA targets, and their corresponding crRNA are shown in Table 5 below.
  • competent cells 25 ⁇ of E. coli DH10B were electroporated with 50 ng of a plasmid for expression of Effector with Chloramphenicol resistance gene and 50 ng of a plasmid for expression of crRNA with Spectinomycin resistance gene. Electroporation was performed using ImM cuvettes and the standard Biorad electroporator settings for bacteria (1.8kV, 200mW, 25 ⁇ ). Immediately after electroporation, cells were re-suspended in 900 uL of SOC media and incubated at 37 °C for 1 h.
  • RNA- interference activity was calculated by comparing transformation frequencies of cultures co- expressing effector and crRNA targeting a known E. coli transcript; against co-expressing effector and crRNA not-targeting a known E. coli transcript. The relative reduction in transformation frequency indicates the activity of system for RNA-guided RNA- interference.
  • Figure 15 illustrates the RNA-guided and RNA-interference activity for Es4Mmc2, Es5Mmc2 and EupMmc2 when co-expressed with crRNAs targeting two independent transcripts in E. coli. No reduction in cell viability was observed when Es4Mmc2, Es5Mmc2 and EupMmc2 were co-expressed with a non-cognate or non-targeting crRNAs (Figure 16).
  • HEPN domains are predicted to play critical roles in RNA cleavage reactions.
  • putative HEPN domains were mutated by substituting the conserved Arginine residue of the RxxxxH motif for Alanine (AxxxxH).
  • Two variants dEs4Mmc2 and dEs5Mmc2 were assessed for their RNA-guided RNase activities in vivo and showed complete suppression of the toxicity observed when Wt Mmc2 proteins were co-expressed with a crRNA targeting an essential gene (Figure 22). This data supports the bioinformatic prediction that Mmc2s are HEPN- dependent RNA-interference systems and that HEPN domains are responsible for the target- specific effects in vivo.
  • Engineered Mmc2 systems were tested in vitro for their ability to catalyze RNA-guided cleavage of ssRNA. Assays were performed with three components: 1) purified Mmc2 effector protein, 2) a synthetic processed CRISPR RNA and 3) ssRNA to act as target RNA.
  • RNA samples were prepared by in vitro transcription from dsDNA templates containing SP6 or T7 promoter sequences.
  • the HiScribeTM High Yield RNA Synthesis Kit (NEB E2070S) was used and for templates containing T7 promoter the MegaShortScriptTM T7 Transcription Kit (Thermo Fisher Scientific AM1354) was used following manufacturer protocol. All RNA products were purified using RNA Clean and ConcentratorTM (Zymo Research). Labeled ssRNA were prepared similarly using 1 :3 of cyanine 5-UTP and standard UTP.
  • Assays were performed using 1 ⁇ of effector protein, 100 nM of crRNA and ssRNA in nuclease buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.5 mM MgC12). Enzymatic reactions were incubated at 37 °C for 2 h and cleaned up using RNA Clean and ConcentratorTM kit. Reaction products were analyzed by 1.2% agarose gel electrophoresis or 8% PAGE TBE-Urea.
  • This feature is particularly interesting for applications involving Mmc2 as antimicrobials.
  • delivery of effector and CRISPR array via phage into specified bacteria could be used to eliminate infections without disturbing the native microbial community.
  • the ability to deliver multiple targeting crRNA's in one transcript could overcome the high rates of mutations that allow pathogenic bacteria to escape CRISPR/Cas nucleases.
  • the in vitro data complements the in vivo data that Mmc2 effector proteins cleave ssRNA in a sequence specific manner that is determined by hybridization of the target RNA and to the crRNA of the crRNA-Mmc2 complex.
  • CRISPR/Cas Effectors are capable of processing their cognate CRIPSR array (i.e. multiple modules of direct repeat and spacer sequence), while others can only function in certain heterologous hosts if the correct processed form is co-expressed, e.g. Cas9. For this reason, Mmc2 Effectors were evaluated for their ability to process a cognate CRISPR array.
  • the purified Es4Mmc2 effector protein was incubated with a CRISPR array containing thirteen direct repeats sequences separated by thirteen unique spacer sequences. Analysis of the cleavage products revealed distinct bands at 50nt, 100 nt, 150 nt, 200 nt as predicted to be the size of processed crRNA based on the RNA sequencing analysis ( Figure 20). This shows that Es4Mmc2 Effector protein can process CRISPR array and could possibly be used for multiplex engineering transcriptomes by recognition of multiple targets. This feature is particularly interesting for applications involving Mmc2 as antimicrobials where utilization of multiple targets could overcome the high rates of mutations that allow pathogenic bacteria to escape CRISPR/Cas nucleases.
  • HEPN mutants were also evaluated for their ability to process multiplexed crRNAs in vivo.
  • Small RNAs were extracted and sequenced from E. coli cultures co-expressing HEPN mutants of dEs5Mmc2s mutants and a minimal CRISPR array containing direct repeat-spacer-direct repeat-spacer. Analysis of RNA sequencing data revealed that Mmc2 effectors comprising the mutant HEPN domain can still process CRISPR array and generate multiple crRNAs in vivo ( Figure 8).
  • Mmc2 systems comprising mutant Mmc2 effectors that are unable to cleave a target RNA, can still bind to the target RNA through crRNA.
  • Mmc2 effector protein can be fused to one or more Nuclear Localization Signal (NLS) in the N-terminal and/or C- terminal. Additionally, inactive versions of Mmc2 can be further fused to RNA editing enzymes for modification of one or multiple RNA target sites.
  • NLS Nuclear Localization Signal
  • Mmc2s In vitro assays were performed to verify whether Mmc2s also exhibit a collateral effect.
  • the assay utilized four components: 1) purified Mmc2 effector protein, 2) a synthetic processed CRISPR RNA, 3) ssRNA to act as target and 4) a labeled ssRNA with distinct sequence from the target ssRNA. Degradation of the labeled non-target ssRNAs is only observed when Mmc2 effector protein was incubated with crRNA containing spacer sequence complementary to the target ssRNA (non-labeled). No degradation of the labeled ssRNAs is observed if the Mmc2 effector protein is incubated with a non-complementary crRNA or if the target ssRNA is missing.
  • Es4Mmc2 and Es5Mmc2 effectors only exhibit collateral effect upon interaction of the effector protein, crRNA and target ssRNA ( Figure 22).
  • Mmc2 CRISPR systems can be introduced into prokaryotic or eukaryotic cell to perturb the cellular gene transcripts.
  • the perturbation can be gene specific using target specific crRNA.
  • the perturbation can be non-specific due to the collateral effect.
  • the Mmc2 effectors may comprise LS at their N- or C- terminus.
  • Mmc2 CRISPR systems can be introduced into cancer cells.
  • the RNA inside the cancer cells can be targeted by expressing two components of the Mmc2 CRISPR system in the cancer cells: a) Mmc2 effector and b) multiple crRNA targeting transcripts that are specific to cancerous cells.
  • the nonspecific RNase activity can be activated and initiates apoptosis of cancerous cells.
  • RNA viruses can be used for detection of non-specific RNase activity upon activation of Mmc2 effector protein in the presence of a crRNA designed to target a transcript of interest.
  • RNA viruses as example Ebola, Zika, Dengue.
  • this technology can also be used for detection of bacterial pathogens or identification of transcripts associated with specific cancer cells.
  • Catalytic inactive versions Mmc2 CRISPR systems i. e. HEPN mutants
  • the Mmc2 effectors may be fused to an RNA editing enzyme and an NLS at their N- or C- terminus.
  • mammalian RNA editing enzymes are Adenosine Deaminases Acting on RNA enzymes (ADARs) whose abnormal activities are associated with a wide range of human diseases.
  • ADARs naturally deaminates dsRNA so for this purpose the Mmc2 system can be engineered to create a site-specific dsRNA structure via hybridization of crRNA to the target ssRNA that can be edited by ADARs.
  • the Casl3d protein was selected from Ruminococcus flavefaciens XPD3002 and designed to carry a nuclear localization signal from SV40 as well as a flag-tag to check for proper expression within the host.
  • the Mmc2- LS-Flagged gene was then codon optimized using SGI proprietary software, Archetype, to match Nannochloropsis gaditana (STR00002) codon usage.
  • the gene was put under the control of the Nitrite Reductase (EMRE2EUKT373295) promoter and terminator which is tightly repressed by ammonia.
  • This gene/promoter/terminator set was cloned into a plasmid containing constitutive GFP and bleomycin resistance gene.
  • the plasmid was cut to release and linearize the GFP/Mmc2/BleomycinR fragment which was transformed into Nannochloropsis via electroporation with selection on zeocin. Colonies from this transformation were patched out and GFP expression was assessed to indicate the proper integration of the gene fragments. These colonies are scheduled to assess protein expression using Western blot with an anti-flag antibody to be used as a background strain to test in vivo guide RNA nuclease activity.
  • the guide RNAs were designed for two separate targets.
  • One target Signal Recognition Peptide 54 (SRP54) upon attenuation or disruption will reduce pigment levels allowing for assessment of activity via visible phenotype.
  • Another target Nitrate Reductase, is required for utilization of nitrate as a nitrogen source and will cause death if disrupted.
  • the guide RNAs were designed to be transcriptionally controlled using the Nitrite Reductase promoter/terminator pair. This regulation will allow for ammonia to repress both the Mmc2 effector as well as the guide transcription, while switching to nitrate will induce Mmc2 expression as well as guide transcription inducing cleavage of mRNAs targeted.
  • the guides were designed as mature guides with 20bp targeting sequence to either gene of interest.
  • the guides were flanked with self cleaving ribozymes to release the guide RNAs from the adjacent transcript and UTR regions.

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Abstract

La présente invention concerne des systèmes, des procédés, des kits et des compositions de modification d'ARN utilisant de nouveaux effecteurs Mmc2. Les polypeptides Mmc2 fonctionnent comme des effecteurs de type VI de classe 2, et ont une activité endonucléase d'ARN. Les polypeptides, les acides nucléiques, les vecteurs d'expression, les cellules hôtes et les procédés selon la présente invention ont des applications dans de nombreux domaines y compris, par exemple, la biologie synthétique, les outils de biologie moléculaire, le traitement d'êtres humains et d'animaux, et le diagnostic.
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US11005799B2 (en) 2017-08-22 2021-05-11 Salk Institute For Biological Studies RNA targeting methods and compositions
US11025574B2 (en) 2017-08-22 2021-06-01 Salk Institute For Biological Studies RNA targeting methods and compositions
US11032224B2 (en) 2017-08-22 2021-06-08 Salk Institute For Biological Studies RNA targeting methods and compositions
US11032225B2 (en) 2017-08-22 2021-06-08 Salk Institute For Biological Studies RNA targeting methods and compositions
EP3673055A4 (fr) * 2017-08-22 2021-08-11 Salk Institute for Biological Studies Méthodes et compositions de ciblage d'arn
WO2019040664A1 (fr) 2017-08-22 2019-02-28 Salk Institute For Biological Studies Méthodes et compositions de ciblage d'arn
US11970720B2 (en) 2017-08-22 2024-04-30 Salk Institute For Biological Studies RNA targeting methods and compositions
US11228547B2 (en) 2017-08-22 2022-01-18 Salk Institute For Biological Studies RNA targeting methods and compositions
US11316812B2 (en) 2017-08-22 2022-04-26 Salk Institute For Biological Studies RNA targeting methods and compositions
US11303593B2 (en) 2017-08-22 2022-04-12 Salk Institute For Biological Studies RNA targeting methods and compositions
US11303592B2 (en) 2017-08-22 2022-04-12 Salk Institute For Biological Studies RNA targeting methods and compositions
US11310180B2 (en) 2017-08-22 2022-04-19 Salk Institute For Biological Studies RNA targeting methods and compositions
US11310179B2 (en) 2017-08-22 2022-04-19 Salk Institute For Biological Studies RNA targeting methods and compositions
WO2021016453A1 (fr) * 2019-07-23 2021-01-28 University Of Rochester Clivage d'arn ciblé avec crispr-cas
EP4065703A4 (fr) * 2019-11-26 2024-09-25 New York Genome Center Inc Méthodes et compositions impliquant des guides de classe 2, de type vi, de crisp
WO2021195519A1 (fr) * 2020-03-27 2021-09-30 University Of Rochester Destruction ciblée d'arn viral par crispr-cas13
WO2021195525A1 (fr) * 2020-03-27 2021-09-30 University Of Rochester Réseaux d'arncr crispr-cas13
WO2023096584A3 (fr) * 2021-11-25 2023-06-22 Casbio (S) Pte Ltd Nouveaux systèmes crispr/cas13 et leurs utilisations

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