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WO2022188039A1 - Système crispr/cas13 modifié et ses utilisations - Google Patents

Système crispr/cas13 modifié et ses utilisations Download PDF

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WO2022188039A1
WO2022188039A1 PCT/CN2021/079821 CN2021079821W WO2022188039A1 WO 2022188039 A1 WO2022188039 A1 WO 2022188039A1 CN 2021079821 W CN2021079821 W CN 2021079821W WO 2022188039 A1 WO2022188039 A1 WO 2022188039A1
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Prior art keywords
cas13
sequence
engineered
rna
residues
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Huawei TONG
Shaoran Wang
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Huigene Therapeutics Co Ltd
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Huigene Therapeutics Co Ltd
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Priority to PCT/CN2021/079821 priority Critical patent/WO2022188039A1/fr
Priority to PCT/CN2021/121926 priority patent/WO2022068912A1/fr
Priority to CN202180018124.0A priority patent/CN116096875B/zh
Priority to CN202410088378.5A priority patent/CN118109438A/zh
Priority to EP21793851.3A priority patent/EP4222253A1/fr
Priority to EP22710479.1A priority patent/EP4305157A1/fr
Priority to CN202280003194.3A priority patent/CN115427561B/zh
Priority to PCT/CN2022/079890 priority patent/WO2022188797A1/fr
Priority to US17/836,175 priority patent/US20220389398A1/en
Priority to US17/836,266 priority patent/US20230075045A1/en
Publication of WO2022188039A1 publication Critical patent/WO2022188039A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • CRISPR clustered regularly interspaced short palindromic repeats
  • prokaryotic organisms such as bacteria and archaea. These sequences are understood to be derived from DNA fragments of bacteriophages that have previously infected the prokaryote, and are used to detect and destroy DNA or RNA from similar bacteriophages during subsequent infections of the prokaryotes.
  • CRISPR-associated system is a set of homologous genes, or Cas genes, some of which encode Cas protein having helicase and nuclease activities.
  • the Cas proteins are enzymes that utilize RNA derived from the CRISPR sequences (crRNA) as guide sequences to recognize and cleave specific strands of polynucleotide (e.g., DNA) that are complementary to the crRNA.
  • crRNA CRISPR sequences
  • the CRISPR-Cas system constitutes a primitive prokaryotic “immune system” that confers resistance or acquired immunity to foreign pathogenic genetic elements, such as those present within extrachromosomal DNA (e.g., plasmids) and bacteriophages, or foreign RNA encoded by foreign DNA.
  • extrachromosomal DNA e.g., plasmids
  • bacteriophages e.g., bacteriophages
  • CRISPR/Cas system appears to be a widespread prokaryotic defense mechanism against foreign genetic materials, and is found in approximately 50%of sequenced bacterial genomes and nearly 90%of sequenced archaea.
  • This prokaryotic system has since been developed to form the basis of a technology known as CRISPR-Cas that found extensive use in numerous eukaryotic organisms including human, in a wide variety of applications including basic biological research, development of biotechnology products, and disease treatment.
  • the prokaryotic CRISPR-Cas systems comprise an extremely diverse group of effector proteins, non-coding elements, as well as loci architectures, some examples of which have been engineered and adapted to produce important biotechnologies.
  • the CRISPR locus structure has been studied in many systems.
  • the CRISPR array in the genomic DNA typically comprises an AT-rich leader sequence, followed by short DR sequences separated by unique spacer sequences.
  • These CRISPR DR sequences typically range in size from 28 to 37 bps, though the range can be 23-55 bps.
  • Some DR sequences show dyad symmetry, implying the formation of a secondary structure such as a stem- loop ( “hairpin” ) in the RNA, while others appear unstructured.
  • the size of spacers in different CRISPR arrays is typically 28-38 bps (with a range of 21-72 bps) . There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.
  • cas genes are often found next to such CRISPR repeat-spacer arrays. So far, the 93 identified cas genes have been grouped into 35 families, based on sequence similarity of their encoded proteins. Eleven of the 35 families form the so-called cas core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the cas core.
  • CRISPR-Cas systems can be broadly divided into two classes -Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids, while Class 2 systems use a single large Cas protein for the same purpose.
  • Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids
  • Class 2 systems use a single large Cas protein for the same purpose.
  • the single-subunit effector compositions of the Class 2 systems provide a simpler component set for engineering and application translation, and has thus far been important sources of discovery, engineering, and optimization of novel powerful programmable technologies for genome engineering and beyond.
  • Class 1 system is further divided into types I, III, and IV; and Class 2 system is divided into types II, V, and VI. These 6 system types are additionally divided into 19 subtypes. Classification is also based on the complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1 protein. Many prokaryotes contain multiple CRISPR-Cas systems, suggesting that they are compatible and may share components.
  • Cas9 is a DNA endonuclease activated by a small crRNA molecule that complements a target DNA sequence, and a separate trans-activating CRISPR RNA (tracrRNA) .
  • the crRNA consists of a direct repeat (DR) sequence responsible for protein binding to the crRNA and a spacer sequence, which may be engineered to be complementary to any desired nucleic acid target sequence. In this way, CRISPR systems can be programmed to target DNA or RNA targets by modifying the spacer sequence of the crRNA.
  • DR direct repeat
  • sgRNA single guide RNA
  • sgRNA single guide RNA
  • Cas9 effector protein from other species have also been identified and used similarly, including Cas9 from the S. thermophilus CRISPR system.
  • CRISPR/Cas9 systems have been widely used in numerous eukaryotic organisms, including baker’s yeast (Saccharomyces cerevisiae) , the opportunistic pathogen Candida albicans, zebrafish (Danio rerio) , fruit flies (Drosophila melanogaster) , ants (Harpegnathos saltator and Ooceraea biroi) , mosquitoes (Aedes aegypti) , nematodes (Caenorhabditis elegans) , plants, mice, monkeys, and human embryos.
  • Cas12a Another recently characterized Cas effector protein is Cas12a (formerly known as Cpf1) .
  • Cas12a together with C2c1 and C2c3, are members belonging to Class 2, type V Cas proteins that lack HNH nuclease, but have RuvC nuclease activity.
  • Cas12a which was initially characterized in the CRISPR/Cpf1 system of the bacterium Francisella novicida. Its original name reflects the prevalence of its CRISPR-Cas subtype in the Prevotella and Francisella lineages.
  • Cas12a showed several key differences from Cas9, including: causing a “staggered” cut in double stranded DNA as opposed to the “blunt” cut produced by Cas9, relying on a “T rich” PAM sequence (which provides alternative targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) and no tracrRNA for successful targeting.
  • Cas12a small crRNAs are better suited than Cas9 for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9’s sgRNAs. Further, the sticky 5’ overhangs left by Cas12a can be used for DNA assembly that is much more target-specific than traditional Restriction Enzyme cloning.
  • Cas12a cleaves DNA 18-23 base pairs downstream from its PAM site, which means no disruption to the nuclease recognition sequence after DNA repair following the creation of double stranded break (DSB) by the NHEJ system, thus Cas12a enables multiple rounds of DNA cleavage, as opposed to the likely one round after Cas9 cleavage because the Cas9 cleavage sequence is only 3 base pairs upstream of the PAM site, and the NHEJ pathway typically results in indel mutations which destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage is associated with an increased chance for the desired genomic editing to occur.
  • Cas13 also known as C2c2
  • Cas13b also known as C2c2
  • Cas13c including the engineered variant CasRx
  • Cas13e and Cas13f
  • RNAi RNA-guided RNase
  • the CRISPR/Cas13 systems can achieve higher RNA digestion efficiency compared to the traditional RNAi and CRISPRi technologies, while simultaneously exhibiting much less off-target cleavage compared to RNAi.
  • CRISPR-Cas13 is quickly becoming a widely adopted RNA editing technology.
  • This system can use its sequence specific guide RNA to selectively modify (e.g., cut or cleave via endonuclease activity) a target RNA, such as mRNA.
  • RNA controls gene expression at the transcription level, thus providing a safer and more controllable gene therapy approach.
  • RNA editing efficiency of the CRISPR/Cas13 systems have already been widely used in a number of organisms including yeast, plant, mammal, and zebra fish (see (Abudayyeh et al., 2017; Aman et al., 2018; Cox et al., 2017; Jing et al., 2018; Konermann et al., 2018) .
  • Cas13 proteins have non-specific /collateral RNase activity upon activation by crRNA-based target sequence recognition. This activity is particularly strong in Cas13a and Cas13b, and still detectably exists in Cas13d and, to a lesser extent, in Cas13e, for example. While this property can be advantageously used in nucleic acid detection methods, the non-specific /collateral RNase activity of these Cas13 proteins also causes undesirable collateral degradation of bystander RNAs, and has imposed a major barrier for their in vivo application, such as in gene therapy.
  • One aspect of the invention provides an engineered Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -Cas13 effector enzyme, wherein the engineered Cas13: (1) comprises a mutation in a region spatially close to an endonuclease catalytic domain (e.g., a HEPN domain) of the corresponding wild-type Cas13 effector enzyme; (2) substantially preserves (e.g., retains at least 50%, 60%, 70%, 72.5%, 75%, 80%, 85%, 87.5%, 90%, 95%, 96%, 97%, 97.5%, 98%, 99%or more of) guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards a target RNA complementary to the guide sequence; and, (3) substantially lacks (e.g., retains less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%,
  • the Cas13 is a Cas13a, a Cas13b, a Cas13c, a Cas13d (including CasRx) , a Cas13e, or a Cas13f.
  • the Cas13e has the amino acid sequence of SEQ ID NO: 4, and/or wherein the Cas13d has the amino acid sequence of SEQ ID NO: Cas13d.
  • the region includes residues within 130, 125, 120, 110, 100, 90, or 80 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXXH domain) in the primary sequence of the Cas13e, and residues within 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXH domain) in the primary sequence of the Cas13d.
  • the endonuclease catalytic domain e.g., an RXXXXH domain
  • the region includes residues more than 100, 110, 120, or 130 residues away from any residues of the endonuclease catalytic domain in the primary sequence of the Cas13, but are spatially within 1-10 or 5 of a residue of the endonuclease catalytic domain.
  • the endonuclease catalytic domain is a HEPN domain, optionally a HEPN domain comprising an RXXXXH motif.
  • the RXXXXH motif comprises a R ⁇ N/H/K/Q/R ⁇ X 1 X 2 X 3 H sequence.
  • X 1 is R, S, D, E, Q, N, G, or Y
  • X 2 is I, S, T, V, or L
  • X3 is L, F, N, Y, V, I, S, D, E, or A.
  • the RXXXXH motif is an N-terminal RXXXXH motif comprising an RNXXXH sequence, such as an RN ⁇ Y/F ⁇ ⁇ F/Y ⁇ SH sequence.
  • the N-terminal RXXXXH motif has a RNYFSH sequence.
  • the N-terminal RXXXXH motif has a RNFYSH sequence.
  • the RXXXXH motif is a C-terminal RXXXXH motif comprising an R ⁇ N/A/R ⁇ ⁇ A/K/S/F ⁇ ⁇ A/L/F ⁇ ⁇ F/H/L ⁇ H sequence.
  • the C-terminal RXXXXH motif has a RN (A/K) ALH sequence.
  • the C-terminal RXXXXH motif has a RAFFHH or RRAFFH sequence.
  • said region comprises, consists essentially of, or consists of: (i) residues corresponding to residues between residues 1-194, 2-187, 227-242, 620-775, or 634-755 of SEQ ID NO: 4; or, (ii) residues corresponding to the HEPN1-1 domain (e.g., residues 90-292) , Helical2 domain (e.g., residues 536-690) , and the HEPN2 domain (e.g., residues 690-967) of SEQ ID NO: Cas13d.
  • said region comprises, consists essentially of, or consists of residues corresponding to residues between residues 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO: 4.
  • said mutation comprises, consists essentially of, or consists of substitutions, within a stretch of 15-20 consecutive amino acids within the region, (a) one or more charged, nitrogen-containing side chain group, bulky (such as F or Y) , aliphatic, and/or polar residues to a charge-neutral short chain aliphatic residue (such as A, V, or I) ; (b) one or more I/L to A substitution (s) ; and/or (c) one or more A to V substitution (s) .
  • said stretch is about 16 or 17 residues.
  • substantially all, except for up to 1, 2, or 3, charged and polar residues within the stretch are substituted.
  • a total of about 7, 8, 9, or 10 charged and polar residues within the stretch are substituted.
  • the N-and C-terminal 2 residues of the stretch are substituted to amino acids the coding sequences of which contain a restriction enzyme recognition sequence.
  • the N-terminal two residues are VF, and the C-terminal 2 residues are ED, and the restriction enzyme is BpiI.
  • the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, and T residues.
  • the one or more charged or polar residues comprise R, K, H, N, Y, and/or Q residues.
  • one or more Y residue (s) within said stretch is substituted.
  • said one or more Y residues (s) correspond to Y672, Y676, and/or Y715 of wild-type Cas13e. 1 (SEQ ID NO: 4) .
  • said stretch is residues 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO: 4.
  • the mutation comprises Ala substitution (s) corresponding to any one or more of SEQ ID NOs: 37-39, 45, and 48.
  • the charge-neutral short chain aliphatic residue is Ala (A) .
  • said mutation comprises, consists essentially of, or consists of: (a) substitutions within 1, 2, 3, 4, or 5 of said stretches of 15-20 consecutive amino acids within the region; (b) a mutation corresponds to a Cas13d mutation of Example 4 that retains at least about 75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d) , and exhibits less than about 27.5%collateral effect of wild-type Cas13d (such as SEQ ID NO: Cas13d) ; (c) a mutation corresponds to the N1V7, N2V7, N2V8 (cfCas13d) , N3V7, or N15V4 mutation of Cas13d mutation; (d) a mutation corresponds to a Cas13d mutation of Example 4 that retains between about 25-75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d) , and exhibit
  • the engineered Cas13 preserves at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA.
  • the engineered Cas13 lacks at least about 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or 100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.
  • the engineered Cas13 preserves at least about 80-90%of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA, and lacks at least about 95-100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.
  • the engineered Cas13 of the invention has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.86%identical to any one of SEQ ID NOs: 6-10 and Cas13d, excluding any one or more of the regions defined by SEQ ID NOs: 16, 20, 24, 28, and 32, and any of the mutation regions in Example 4 or 5.
  • said amino acid sequence contains up to 1, 2, 3, 4, or 5 differences (a) in each of one or more regions defined by SEQ ID NO: 16, 20, 24, 28, and 32, as compared to SEQ ID NOs: 17, 21, 25, 29, and 33, respectively, or (b) in any of the desired mutations in Cas13d and Cas13e disclosed herein.
  • the engineered Cas13 of the invention has the amino acid sequence of any one of SEQ ID NOs: 6-10.
  • the engineered Cas13 of the invention has the amino acid sequence of SEQ ID NO: 9 or 10.
  • the engineered Cas13 of the invention further comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) .
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the engineered Cas13 comprises an N-and/or a C-terminal NLS.
  • Another aspect of the invention provides a polynucleotide encoding the engineered Cas13 of the invention.
  • the polynucleotide of the invention is codon-optimized for expression in a eukaryote, a mammal, such as a human or a non-human mammal, a plant, an insect, a bird, a reptile, a rodent (e.g., mouse, rat) , a fish, a worm /nematode, or a yeast.
  • a mammal such as a human or a non-human mammal
  • a plant an insect, a bird, a reptile, a rodent (e.g., mouse, rat) , a fish, a worm /nematode, or a yeast.
  • a polynucleotide having (i) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotides additions, deletions, or substitutions compared to the polynucleotide of the invention; (ii) at least 50%, 60%, 70%, 80%, 90%, 95%, or 97%sequence identity to the polynucleotide of the invention; (iii) hybridize under stringent conditions with the polynucleotide of the invention, or any of (i) and (ii) ; or (iv) is a complement of any of (i) - (iii) .
  • one or more e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
  • Another aspect of the invention provides a vector comprising the polynucleotide of the invention.
  • the polynucleotide is operably linked to a promoter and optionally an enhancer.
  • the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue specific promoter.
  • the vector is a plasmid.
  • the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex viral (HSV) vector, an AAV vector, or a lentiviral vector.
  • the AAV vector is a recombinant AAV vector of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV 11, AAV 12, or AAV 13.
  • Another aspect of the invention provides a delivery system comprising (1) a delivery vehicle, and (2) the engineered Cas13 of the invention, the polynucleotide of the invention, or the vector of the invention.
  • the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.
  • Another aspect of the invention provides A cell or a progeny thereof, comprising the engineered Cas13 of the invention, the polynucleotide of the invention, or the vector of the invention.
  • the cell or progeny thereof is a eukaryotic cell (e.g., a non-human mammalian cell, a human cell, or a plant cell) or a prokaryotic cell (e.g., a bacteria cell) .
  • a eukaryotic cell e.g., a non-human mammalian cell, a human cell, or a plant cell
  • a prokaryotic cell e.g., a bacteria cell
  • Another aspect of the invention provides A non-human multicellular eukaryote comprising the cell of the invention.
  • the non-human multicellular eukaryote is an animal (e.g., rodent or primate) model for a human genetic disorder.
  • Another aspect of the invention provides A method of modifying a target RNA, the method comprising contacting the target RNA with a CRISPR-Cas13 complex comprising the engineered Cas13 of the invention, and a spacer sequence complementary to at least 15 nucleotides of the target RNA; wherein upon binding of the complex to the target RNA through the spacer sequence, engineered Cas13 modifies the target RNA.
  • the target RNA is modified by cleavage by the engineered Cas13.
  • the target RNA is an mRNA, a tRNA, an rRNA, a non-coding RNA, an lncRNA, or a nuclear RNA.
  • the engineered Cas13 upon binding of the complex to the target RNA, does not exhibit substantial (or detectable) collateral RNase activity.
  • the target RNA is within a cell.
  • the cell is a cancer cell.
  • the cell is infected with an infectious agent.
  • the infectious agent is a virus, a prion, a protozoan, a fungus, or a parasite.
  • the cell is a neuronal cell (e.g., astrocyte, glial cell (e.g., Muller glia cell, oligodendrocyte, ependymal cell, Schwan cell, NG2 cell, or satellite cell) ) .
  • glial cell e.g., Muller glia cell, oligodendrocyte, ependymal cell, Schwan cell, NG2 cell, or satellite cell
  • the CRISPR-Cas13 complex is encoded by a first polynucleotide encoding the engineered Cas13 of the invention, and a second polynucleotide comprising or encoding a spacer RNA capable of binding to the target RNA, wherein the first and the second polynucleotides are introduced into the cell.
  • the first and the second polynucleotides are introduced into the cell by the same vector.
  • the method causes one or more of: (i) in vitro or in vivo induction of cellular senescence; (ii) in vitro or in vivo cell cycle arrest; (iii) in vitro or in vivo cell growth inhibition and/or cell growth inhibition; (iv) in vitro or in vitro induction of anergy; (v) in vitro or in vitro induction of apoptosis; and (vi) in vitro or in vitro induction of necrosis.
  • Another aspect of the invention provides a method of treating a condition or disease in a subject in need thereof, the method comprising administering to the subject a composition comprising a CRISPR-Cas complex comprising the engineered Cas13 of the invention or a polynucleotide encoding the same; and a spacer sequence complementary to at least 15 nucleotides of a target RNA associated with the condition or disease; wherein upon binding of the complex to the target RNA through the spacer sequence, the engineered Cas13 cleaves the target RNA, thereby treating the condition or disease in the subject.
  • condition or disease is a neurological condition, a cancer or an infectious disease.
  • the cancer is Wilms’ tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer.
  • the neurological condition is glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, Leber’s hereditary optic neuropathy, a neurological condition associated with degeneration of RGC neurons, a neurological condition associated with degeneration of functional neurons in the striatum of a subject in need thereof, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Schizophrenia, depression, drug addiction, movement disorder such as chorea, choreoathetosis, and dyskinesias, bipolar disorder, Autism spectrum disorder (ASD) , or dysfunction.
  • Parkinson’s disease Alzheimer’s disease
  • Huntington’s disease Huntington’s disease
  • Schizophrenia depression
  • depression drug addiction
  • movement disorder such as chorea, choreoathetosis, and dyskinesias
  • bipolar disorder Autism spectrum disorder (ASD)
  • ASSD Autism spectrum disorder
  • the method is an in vitro method, an in vivo method, or an ex vivo method.
  • a CRISPR-Cas complex comprising the engineered Cas13 of the invention, a guide RNA comprising a DR sequence that binds the engineered Cas13 and a spacer sequence designed to be complementary to and binds a target RNA.
  • the target RNA is encoded by a eukaryotic DNA.
  • the eukaryotic DNA is a non-human mammalian DNA, a non-human primate DNA, a human DNA, a plant DNA, an insect DNA, a bird DNA, a reptile DNA, a rodent DNA, a fish DNA, a worm /nematode DNA, a yeast DNA.
  • the target RNA is an mRNA.
  • the CRISPR-Cas complex further comprises a target RNA comprising a sequence capable of hybridizing to the spacer sequence.
  • Another aspect of the invention provides a method of identifying an engineered CRISPR/Cas effector enzyme of a corresponding wild-type Cas effector enzyme, wherein the engineered Cas substantially maintains guide-sequence-specific endonuclcase activity and substantially lacks guide-sequence-independent collateral endonuclease activity, the method comprising: (1) in each of one or more regions of 15-20 consecutive polynucleotides (a) within 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 residues of any residues of a endonuclease catalytic domain of the wild-type Cas effector enzyme or (b) spatially within 1-10 of any residues of the endonuclease catalytic domain of the wild-type Cas effector enzyme, substituting one or more (e.g., substantially all, except for up to 1, 2, 3, 4, or 5) polar and charged residues with a charge neutral aliphatic side-chain residue (such as A)
  • the wild-type Cas effector enzyme is a Cas13.
  • the Cas13 is a Cas13a, a Cas13b, a Cas13c, a Cas13d (e.g., CasRx) , a Cas13e, or a Cas13f.
  • the Cas13e has the amino acid sequence of SEQ ID NO: 4; or wherein the Cas13d has the amino acid sequence of SEQ ID NO: Cas13d.
  • Another aspect of the invention provides a method of identifying an engineered Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -Cas13 effector enzyme with altered guide sequence-independent collateral nuclease activity, the method comprising: in a region spatially close to an endonuclease catalytic domain of the corresponding wild-type Cas13 effector enzyme, substituting one or more charged or polar residues to a charge-neutral short chain aliphatic residue (such as A) , to determine whether the resulting variant Cas13 effector enzyme: (1) has substantially preserved guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards a target RNA complementary to the guide sequence; and, (2) either substantially lacks or has enhanced guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards a non-target RNA that does not bind to the guide sequence, thereby identifying said engineered Cas13 effector enzyme with altered guide sequence
  • the engineered Cas13 effcctor enzyme substantially lacks guide sequence-independent collateral nuclease activity.
  • the engineered Cas13 effector enzyme has enhanced guide sequence-independent collateral nuclease activity.
  • said one or more charged or polar residues are within a stretch of 15-20 (e.g., 16 or 17) consecutive amino acids within the region.
  • said one or more charged or polar residues comprise, consist essentially of, or consist of one or more (or all) Tyr (Y) residue (s) within the stretch.
  • FIG. 1 is a schematic (not to scale) illustration of a possible mechanism of reduced collateral effect by a Cas13 (e.g., Cas13e) effector enzyme.
  • the upper left panel shows a possible mechanism of sequence-specific targeting and cleavage of a target RNA by wild-type Cas13e.
  • the upper right panel shows a possible mechanism of non-sequence-specific targeting and cleavage of non-target RNA by wild-type Cas13e.
  • the lower left panel shows a possible mechanism of action by a subject engineered Cas13e with reduced affinity for non-target RNA and higher tendency to cleave target RNA in a sequence-specific manner.
  • FIG. 2 shows a predicted 3D structure of a Cas13e protein.
  • FIG. 3 shows the locations of the mutations in the engineered Cas13e mapped to the wild-type Cas13e sequence.
  • the two HEPN sequences (HEPN1 and HEPN2) are also shown.
  • FIG. 4 is a schematic drawing (not to scale) of the double-fluorescent vector used to identify the subject engineered Cas13e effector proteins.
  • the guide RNA (gRNA) encoded by the vector targets an EGFP reporter. Boxes with dashed lines include the two HEPN RXXXXH sequences (HEPN1 and HEPN2) and their respective nearby sequences (residues 2-187 and 634-755) , as well as a sequence (residues 227-242) predicted to be spatially close to the HEPN sequences in Cas13e. Mutations with desired functional changes in those regions were identified in engineered Cas13e.
  • FIG. 5 shows the relative fluorescent intensity distribution among the various engineered Cas13e effector enzymes (Mut-1 to Mut-21) and Cas13e wild-type positive and negative controls, each shown as the intensity difference between the targeted (guide sequence-specific cleavage of) EGFP signal (left panel) and the control mCherry signal (right panel) .
  • FIG. 6 shows the relative percentage of mCherry positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to 100%relative percentage of mCherry positive cells have no or nearly no non-sequence-specific endonuclease activity, like dCas13e (which has neither sequence-specific nor non-sequence-specific endonuclease activity) .
  • FIG. 7 shows the relative percentage of EGFP positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to wild-type Cas13e relative percentage (e.g., about 20%) of EGFP positive cells have comparable level of sequence-specific endonuclease activity as wild-type Cas13e.
  • FIG. 8 shows the spacial distribution of the various mutations with reduced collateral effect, in the predicted Cas13e 3D structure.
  • FIG. 9 shows the sequences of several mutations in the Mut-17 region.
  • FIG. 10 shows the relative percentage of mCherry positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to 100%relative percentage of mCherry positive cells have no or nearly no non-sequence-specific endonuclease activity, like dCas13e (which has neither sequence-specific nor non-sequence-specific endonuclease activity) .
  • FIG. 11 shows the relative percentage of EGFP positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to wild-type Cas13e relative percentage (e.g., about 20%) of EGFP positive cells have comparable level of sequence-specific endonuclease activity as wild-type Cas13e.
  • FIG. 12 shows the sequences of the rnutations in the Mut-19 region.
  • FIG. 13 shows the relative percentage of mCherry positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to 100%relative percentage of mCherry positive cells have no or nearly no non-sequence-specific endonuclease activity, like dCas13e (which has neither sequence-specific nor non-sequence-specific endonuclease activity) .
  • M17.15-1 and M17.15-2 are the same, and are both double mutants with both Y-to-A mutations in M17.8 and M17.9 (see FIG. 9) .
  • FIG. 14 shows the relative percentage of EGFP positive cells, upon comparing the various engineered Cas13e effector enzymes to wild-type or dCas13e (nuclease null mutant) , after activating Cas13e nuclease activity using guide-sequence specific cleavage of EGFP.
  • Engineered Cas13e effector enzymes with close to wild-type Cas13e relative percentage (e.g., about 20%) of EGFP positive cells have comparable level of sequence-specific endonuclease activity as wild-type Cas13e.
  • FIG. 15 is a schematic drawing showing the domain structures for representative Cas13a-Cas13f effector enzymes. The overall sizes, and the locations of the two RXXXXH motifs on each representative member of the representative Cas13 proteins are indicated.
  • FIGs. 16A-16D show the results of evaluating collateral effects in transiently transfected mammalian cell HEK293T using the dual-fluorescence reporter system of the invention.
  • FIG. 16A is a schematic drawing of the mammalian dual-fluorescence reporter system used to evaluate collateral effects induced by Cas13 (Cas13d/Cas13a) -mediated RNA knockdown.
  • the exemplary dual-fluorescence reporter used herein contains one plasmid with coding sequences for Cas13 (with NLS) and EGFP under the transcription control of the strong CAG promoter, and another plasmid with coding sequences for the various gRNA targeting endogenous or exogenous targets (e.g., mCherry, NT, or RPL4, under the transcriptional control of the U6 promoter) and mCherry (under the transcriptional control of the EF1ct promoter) .
  • endogenous or exogenous targets e.g., mCherry, NT, or RPL4, under the transcriptional control of the U6 promoter
  • mCherry under the transcriptional control of the EF1ct promoter
  • HEK293T cells transfected by the dual-fluorescence reporter system plasmids are subjected to FACS analysis for EGFP (non-specific target) and mCherry (specific target) expression 48 hfs post transfection.
  • FIG. 16B shows the bar graphs summarizing the relative knockdown of exogenous gRNA specific target mCherry and exogenous collateral target EGFP transcripts induced by Cas13d (left panel) or Cas13a (middle panel) with three different mCherry gRNAs, as well as the relative knockdown of the endogenous gRNA specific RPL4 and exogenous collateral target EGFP transcripts induced by Cas13d with four different RPL4 gRNAs (right panel) .
  • FIG. 16C shows FACS quantitative analysis of relative percentage of EGFP or mCherry positive cells from these experiments.
  • FIG. 16D shows characteristics collateral effects of Cas13-mediated endogenous transcripts knockdown in HEK293T cells.
  • differential decreases of relative percentage of EGFP or mCherry positive cells were induced by Cas13d targeting PFN1 (left panel) and PKM (right panel) transcript, with four gRNAs each transcript.
  • FIGs. 17A-17I show results of rational mutagenesis of Cas13d to eliminate collateral activity.
  • FIG. 17A is a schematic drawing of the mammalian dual-fluorescence reporter system used to screen on-target interference activity of Cas13 (shown as Cas13d but broadly represent all Cas13, including Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, and Cas13f, etc. ) , with coding sequences for Cas13, EGFP (target in this experiment) , mCherry (collateral target in this experiment) and EGFP gRNA all in one plasmid.
  • Wild-type (wt) Cas13 cleaves the target EGFP mRNA via the gRNA-specific mechanism and the non-target mCherry mRNA via the collateral activity, dCas13 does not cleave either mCherry or EGFP mRNA for lack of endonuclease activity.
  • the subject engineered Cas13 mutants /variants preserved gRNA-specific EGFP cleavage, but lost the collateral activity against mCherry mRNA.
  • FIG. 17B shows a view of the predicted overall structure (by I-TASSER) of the RfxCas13d complex in ribbon representation.
  • RXXXXH of HEPN domains are the catalytic sites.
  • FIG. 17C shows the 21 regions in HEPN1 (including HEPN1-I and HEPN1-II) , HEPN2, Helical2 and partial Helicall domains of Cas13d selected for mutagenesis studies, with each spanning about 36-amino acids.
  • FIG. 17D shows quantification of relative percentage of EGFP or mCherry positive cells among 118 Cas13d mutants targeting EGFP transcript. WT (wild-type Cas13d) and dead Cas13d (dCas13d) as controls, relative percentages of positive cell were all normalized to dCas13d. FiG.
  • FIG. 17E shows quantification of relative percentage of EGFP or mCherry positive cells among Cas13d mutants with different combinations of mutation sites within or nearby N2V7 and N2V8.
  • WT wild-type Cas13d
  • dCas13d dead Cas13d
  • FIG. 17F shows differential changes of relative percentage of mCherry and EGFP positive cells were induced by cfCas13d with EGFP gRNAs in comparison with Cas13d, dCas13d as control.
  • FIG. 17G and 17H show kinetics of in vitro nuclease activity for Cas13 enzymes.
  • FIGs. 18A and 18B show the cartoon view (FIG. 18A) and opposing surface view (FIG. 18B) of the crystal structure of Cas13d, including the catalytic sites of the HEPN domains (labeled by RXXXXH) , and effective mutated sites (labeled by the various NxVy mutations) .
  • FIG. 18C shows mutated sequences of effective variants from Cas13d.
  • FIGs. 19A-19G show results of rational mutagenesis of Cas13e to improve nuclease specificity.
  • FIG. 19A shows a view of the predicted overall structure of the Cas13e complex in ribbon representation. RXXXXH of HEPN domains are catalytic sites.
  • FIG. 19B shows a mutagenesis scheme according to which the HEPN 1 and HEPN2 domains were mainly selected and divided into 21 mutant regions for further subsequent mutagenesis.
  • FIG. 19C shows quantification of relative percentage of EGFP or mCherry positive cells among Cas13e mutants targeting EGFP transcript.
  • FIG. 19D shows quantification of relative percentage of EGFP or mCherry positive cells among Cas13e mutants from different combinations of mutation sites based on M17 targeting EGFP transcript. Cas13e and dCas13e as used as controls.
  • FIG. 19E and 19F show kinetics of in vitro nuclease activity for Cas13 enzymes. In vitro collateral ribonuclease activity (FIG. 19E) analysis and target ribonuclease activity (FIG.
  • FIG. 19F shows differential changes of mCherry and EGFP fluorescence intensity induced by cfCas13e with EGFP gRNAs in comparison with Cas13e.
  • FIGs. 20A-20I show efficient and specific interference activity of cfCas13d targeting endogenous genes in HEK293 cells.
  • FIG. 20A shows relative expression level (as measured by CPM, counts per million) of 23 endogenous genes in HEK293 cells from RNA-seq of dCas13d groups.
  • FIG. 20B shows differential decreases of relative percentage of EGFP or mCherry positive cells induced by Cas13d targeting 22 endogenous transcripts, with 1-7 gRNAs each transcript, compared with NT.
  • FIG. 20C shows statisticalal quantification from FIG. 20B.
  • FIG. 20H shows Cas13d and cfCas13d targeting of 14 endogenous transcripts in HEK293 cells. Transcript levels are relative to dCas13d as vehicle control.
  • FIG. 20I shows statisticalal data analysis from FIG. 20H.
  • NT non- targeting gRNA.
  • FIGs. 20J and 20K show differential gene expression of Cas13d /cfCas13d targeting CA2 /B4GALNT1 transcripts by flow cytometry analysis.
  • FIGs. 21A-21E show the results of transcriptome-wide off-target edits analysis of Cas13d /cfCas13d targeting endogenous transcript.
  • FIG. 21A shows characteristic of gRNA dependent off-target sites from RPL4-g3, PPIA-g1, CA2-g1 or PPARG-g1, measured in Cas13d and cfCas13d groups. MM #, mismatch number of off-target sites.
  • FIG. 21B shows statisticalal data analysis from FIG. 21A, of which off-target sites with one or more mismatches were analyzed.
  • FIGs. 21C-21D show biological process of significant down-regulated genes induced by Cas13d /cfCas13d-mediated RPL4 (FIG.
  • FIGs. 22A-22C show cellular consequences and working model of collateral effects and its elimination.
  • FIG. 22A is a schematic drawing of the dox-inducible Cas13d /cfCas13d /dCas13d expression system with RPL4 gRNA1 used to examine collateral effects. Representative bright-field images of HEK293T cell clones with dox-inducible Cas13d/cfCas13d/dCas13d expression system during 5 days after dox treatment were not shown.
  • FIG. 22B left panel shows relative RPL4 mRNA knockdown by dCas13d/Cas13d/cfCas13d with RPL4 gRNA in the presence or absence of dox during 5 days.
  • FIG. 22C is a model of Cas13 on-target and collateral cleavage activity.
  • cfCas13 e.g., cfCas13d and cfCas13e
  • Two-tailed unpaired two-sample t-test was used for statisticalal analysis. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ns, no significance.
  • FIGs. 23A-23J is an exemplary multi-sequence alignment of several representative Cas13 family proteins (e.g., Cas13b, Cas13e and Cas13f) , and the domain organizations including the HPEN domains.
  • Cas13 family proteins e.g., Cas13b, Cas13e and Cas13f
  • domain organizations including the HPEN domains.
  • FIGs. 24A-24M is an exemplary multi-sequence alignment of several representative Cas13 family proteins (e.g., Cas13d, Cas13a and Cas13c) , and the domain organizations including the HPEN domains.
  • Cas13 family proteins e.g., Cas13d, Cas13a and Cas13c
  • CRISPR-Cas systems A broad range of CRISPR-Cas systems has been discovered, and a classification system and a common nomenclature have been established for the associated Cas genes. Under such classification system, the CRISPR-Cas systems and the associated effector enzymes belong to two classes -Class 1 and Class 2 -each further divided into three types and numerous subtypes based on their signature Cas genes.
  • the Class 1 systems encompass types I, III, and IV systems, utilizing multisubunit RNA-Protein (RNP) complexes.
  • the Class 2 systems encompass types Ii, V, and VI systems, utilizing single protein RNP complexes.
  • Cas9 is a Class 2, type II effector enzyme
  • Cas13 enzymes including Cas13a, Cas13b, Cas13c, Cas13d (including the engineered variant CasRx) , Cas13e, and Cas13f are Class 2, type VI effector enzymes.
  • Class 2 type VI effector proteins Unlike any other CRISPR-Cas systems, Class 2 type VI effector proteins have been demonstrated to exclusively cleave RNA targets.
  • Such Class 2 type VI effector enzymes have two distinct active sites, both conferring RNase activity: one involved in pre-crRNA processing, the other involved in target RNA degradation.
  • Class 2 type VI Several subtypes of Class 2 type VI exist, including at least subtype VI-A (Cas13a/C2c2) , VI-B (Cas13b1 and Cas13b2) , VI-C (Cas13c) , VI-D (Cas13d, CasRx) , VI-E (Cas13e) , and VI-F (Cas13f) .
  • the Cas13 subtypes generally share very low sequence identity /similarity, but can all be classified as type VI Cas proteins (e.g., generally referred to herein as “Cas13” ) based on the presence of two conserved HEPN-like RNase domains. See FIG. 15.
  • Cas13a from Leptotrichia shahii (LshCas13a) , Lachnospiraceae bacterium (LbaCas13a) , and Leptotrichia buccalis (LbuCas13a) .
  • the crRNA-Cas13a complex is bi-lobed with a nuclease (NUC) lobe and a crRNA recognition (REC) lobe.
  • the crRNA-bound form of Cas13a adopts a “clenched fist” -like structure, with the REC lobe being imperfectly stacked on top of the NUC lobe.
  • the REC lobe has a variable N-terminal domain (NTD) , followed by a helical domain (Helical-1) .
  • NTD N-terminal domain
  • Helical-1 a helical domain
  • the NUC lobe consists of the two HEPN domains (HEPN-1 and HEPN-2) separated by a linker domain (Helical-3) .
  • the HEPN-1 domain is split into two subdomains by another helical domain (Helical-2) .
  • the NTD, Helical-l, and HEPN2 domains form a narrow, positively charged cleft that anchors the 5’ repeat-derived end of the bound crRNA (the 5’-handle) , whereas the 3’ end of the crRNA is bound by the Helical-2 domain.
  • the Cas13 CRISPR locus is initially transcribed into a long pre-crRNA transcript.
  • the Cas13 proteins then cleave the pre-crRNA at fixed positions upstream of the stem-loop structure formed by the palindromic nature of the direct repeat (DR) sequences.
  • Pre-crRNA processing in type VI involves metal-independent cleavages upstream of the stem-loop, and does not require a trans-activating crRNA (tracrRNA) or other host factors.
  • the mature crRNA which comprises a DR sequence and a guide sequence complementary to a target RNA, assembles with the Cas13 proteins to form a functional RNP complex, which then scans transcripts for the complementary RNA target. Once such RNA target is found and bound by the guide sequence, the RNA target is degraded by the Cas13 endonuclease.
  • the Cas13 effector enzymes display unprecedented sensitivity to recognize specific target RNAs within a heterogeneous population of non-target RNAs. It has been reported that Cas13 can detect target RNAs with femtomolar sensitivity. Thus on the one hand, the Class 2 type VI enzymes or Cas13 offer tremendous opportunity to knock down target gene products (e.g., mRNA) for gene therapy, yet on the other hand, such use is inherently limited by the co-called collateral activity that poses significant risk of cytotoxicity.
  • target gene products e.g., mRNA
  • a guide sequence non-specific RNA cleavage referred to as “collateral activity” is conferred by the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain in Cas13 after target RNA binding.
  • HEPN eukaryotes and prokaryotes nucleotide-binding
  • Binding of its cognate target ssRNA complementary to the bound crRNA causes substantial conformational changes in Cas13 effector enzyme, leading to the formation of a single, composite catalytic site for guide-sequence independent “collateral” RNA cleavage, thus converting Cas13 into a sequence non-specific ribonuclease.
  • This newly formed highly accessible active site would not only degrade the target RNA in cis if the target RNA is sufficiently long to reach this new active site, but also degrade non-target RNAs in trans based on this promiscuous RNase activity.
  • RNAs appear to be vulnerable to this promiscuous RNAse activity of Cas13, and most (if not all) Cas13 effector enzymes possess this collateral endonuclease activity. It has been shown recently that the collateral effects by Cas13-mediated knockdown exist in mammalian cells and animals (manuscript submitted) , suggesting that clinical application of Cas13-mediated target RNA knock down will face significant challenge in the presence of collateral effect.
  • subtype VI-B systems include a natural means to regulate the collateral activity of Cas13b via the type VI-associated genes csx27 and csx28, but such natural regulatory mechanism appears to be unique to subtype VI-B, as similar mechanism does not seem to exist in other subtypes such as type VI-A and VI-C.
  • Cas13d and Cas13e variants obtained by structure-guided mutagenesis were screened. It was found that several variants with 2-4 mutations on the Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains retained undiminished on-target activity, but greatly reduced collateral effects. For the Cas13d variant with diminished collateral effect, the transcriptome-wide off-target editing and cell growth arrest observed in wild-type Cas13d were eliminated.
  • HEPN Higher Eukaryotes and Prokaryotes Nucleotide-binding
  • Cas13 is believed to contain two separated binding domains proximal to the HEPN domains -one is responsible for on-target cleavage, and both are required for collateral cleavage.
  • the invention described herein provides engineered high-fidelity Class 2 type VI or Cas13 (e.g., Cas13d and Cas13e) effector enzyme variants with minimal residual collateral effects. These variants are useful, for example, in targeting degradation of RNAs in basic research and therapeutic applications.
  • Cas13d and Cas13e engineered high-fidelity Class 2 type VI or Cas13 effector enzyme variants with minimal residual collateral effects.
  • the invention provides engineered Class 2 type VI or Cas13 (e.g., Cas13e) effector enzymes that largely maintain their sequence-specific endonuclease activity against a target RNA, yet with diminished if not eliminated non-guide sequence-specific endonuclease activity against non-target RNAs.
  • engineered Cas13 effector enzymes that substantially lack collateral effect pave the way for using Cas13 in target RNA-knock down-based utility, such as gene therapy.
  • Such engineered Cas13 effector enzymes that substantially lack collateral effect are also useful for RNA-base editing, because a nuclease dead version (or “dCas13” ) of such engineered Cas13 also has reduced off-target effect, which is still present in dCas13 without the mutations in the subject engineered Cas13.
  • dCas13 nuclease dead version
  • FIGs. 1 and 22C provide plausible mechanisms consistent with the data presented herein.
  • a wild-type Cas13 not only possesses the ability to bind a target RNA through the guide sequence of the crRNA, but also possesses a non-specific RNA binding site (see the oval shaped motif around the catalytic site) for any RNA at the vicinity of the HEPN catalytic domains.
  • a conformation change of Cas13 activates its catalytic activity, and the target RNA, bound by both the complementary guide sequence and the non-specific RNA binding site, is cleaved.
  • Cas13 also non-specifically cleave non-target RNA that does not bind to the guide sequence, partly due to the binding of such non-target RNA to the non-specific RNA binding site on cas13. Mutations in the non-specific RNA binding motif (as signified by a different shade of the oval motif) reduces /eliminates (or in some cases enhances) the ability of Cas13 to bind RNA, thus collateral activity against non-target RNA is reduced /eliminated (or enhanced) without significantly affecting target RNA cleavage because the target RNA is still bound by the guide sequence.
  • off-target effect in RNA-base editing using a nuclease-deficient (dCas13) version of the engineered Cas13 can also be reduced or eliminated, because the loss of non-specific RNA binding in the engineered dCas13 reduced /eliminates unintended RNA based editing due to the proximity of the RNA base editing domain (e.g., ADAR or CDAR) and an off-target RNA substrate.
  • dCas13 nuclease-deficient
  • the invention also provides engineered Class 2 type VI or Cas13 (e.g., Cas13d and Cas13e) effector enzymes that largely maintain their sequence-specific endonuclease activity against a target RNA, yet with enhanced non-guide sequence-specific endonuclease activity against non-target RNAs compared to the corresponding wild-type Cas13.
  • engineered Class 2 type VI or Cas13 e.g., Cas13d and Cas13e
  • Such engineered Cas13 with enhanced collateral effect provides a better (e.g., more sensitive) variant, compared to the wild-type, in nucleic acid detection assays such as SHERLOCK, which takes advantage of the collateral activity to provide an extreme sensitive assay for detecting very small quantities of a guide sequence-specific target RNA in a sample, with or without pre-amplification of the initial nucleic acids in the sample.
  • one aspect of the invention provides an engineered Class 2 type VI Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -Cas effector enzyme, such as Cas13 (e.g., Cas13d and Cas13e) wherein the engineered Class 2 type VI Cas effector enzyme: (1) comprises a mutation in a region spatially close to an endonuclease catalytic domain of the corresponding wild-type effector enzyme; (2) substantially preserves guide sequence-specific endonuclease cleavage activity of the wild-type effector enzyme towards a target RNA complementary to the guide sequence; and, (3) either substantially lacks or has enhanced guide sequence-independent collateral endonuclease cleavage activity of the wild-type effector enzyme towards a non-target RNA that is substantially not complement to /does not bind to the guide sequence.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • the mutation occurs within a region, e.g., within one of two RNA binding domains at, near, or proximal to one of the HEPN-type catalytic domains, of a wild-type Cas13 (such as Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f etc) .
  • the mutation weakens (e.g., significantly weakens or eliminates) binding of the wild-type Cas13 to a non-specific RNA target (e.g., one not substantially complementary to a guide RNA) , but substantially retains binding to a target RNA substantially complementary to the guide RNA.
  • the mutation causes steric hindrance effects and/or change in charge, polarity, and/or size of the sidechain of the involved residues, leading to weakened interactions between activated Cas13 and promiscuous RNA, but not much (if any) effect between activated Cas13 and the on-target RNA.
  • Cas13 is a Class 2 type VI CRISPR-Cas effector enzyme that displays collateral activity as wild-type enzyme upon binding to a cognate target RNA complementary to a guide sequence of its crRNA.
  • the collateral activity of a wild-type Class 2 type VI effector enzyme enables it to cleave RNase or endonuclease activity against a non-target RNA that does not or substantially does not complement with the guide sequence of the crRNA.
  • the wild-type Class 2 type VI effector enzyme may also exhibit one or more of the following characteristics: having one or two conserved HEPN-like RNase domains, such as HEPN domains having the conserved RXXXXH motif (with X being any amino acid) , e.g., the RXXXXH motifs described herein below; having a “clenched fist” -like structure when the Class 2 type VI effector enzyme (e.g., Cas13) binds a cognate crRNA; having a bi-lobed structure with a nuclease (NUC) lobe and a crRNA recognition (REC) lobe, optionally, the REC lobe has a variable N-terminal domain (NTD) , followed by a helical domain (Helical-1) , and/or optionally, the N UC lobe consists of the two HEPN domains (HEPN-1 and HEPN-2) separated by a linker domain (Helical-3)
  • the Class 2 type VI effector enzyme e.g., Cas13
  • the Class 2 type VI effector enzyme has one of the RXXXXN motifs in the HEPN-like domains located at or close to (e.g., within 50-160 residues, or within 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 residues of) the N-terminus.
  • the Class 2 type VI effector enzyme e.g., Cas13
  • the Class 2 type VI effector enzyme has one of the RXXXXN motifs in the HEPN-like domains located at or close to (e.g., within 50-160 residues, or within 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 residues of) the C-terminus.
  • the Class 2 type VI effector enzyme (e.g., Cas13) has one of the RXXXXN motifs of the HEPN-like domains located at or close to (e.g., within 50-160 residues, or within 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 residues of) the N-terminus, while the other of the RXXXXN of the HEPN-like domains is located at or close to (e.g., within 50-160 residues, or within 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 residues of) the C-terminus.
  • An RXXXXN motif is “at or near” the N-or C-terminus, if either the R or the N residue of the RXXXXN motif is at or near the N-or C-terminus.
  • the engineered Class 2 type VI effector enzyme e.g., Cas13 particularly Cas13e
  • the engineered Class 2 type VI effector enzymes have drastically reduced non-sequence-specific endonuclease activity against non-target RNAs, yet simultaneously exhibiting substantially the same if not higher sequence-specific endonuclease activity against a target RNA that substantially complements the guide sequence of the crRNA.
  • the engineered effector enzymes enable high fidelity RNA targeting /editing.
  • the Class 2 type VI effector enzyme is Cas13a, Cas13b, Cas13c, Cas13d (including the engineered variant CasRx) , Cas13e, or Cas13f, or an ortholog, paralog, homolog, natural or engineered variant thereof, or functional fragment thereof that substantially maintains the guide sequence-specific endonuclease activity.
  • the variant or functional fragment thereof maintains at least one function of the corresponding wild-type effector enzyme.
  • functions include, but are not limited to, the ability to bind a guide RNA /crRNA of the invention (described herein below) to form a complex, the guide sequence-specific RNase activity, and the ability to bind to and cleave a target RNA at a specific site under the guidance of the crRNA that is at least partially complementary to the target RNA.
  • the Cas13 protein is a Cas13a protein.
  • the Cas13a protein is from a species of the genus Bacteroides, Blautia, Butyrivibrio, Carnobacterium, Chloroflexus, Clostridium, Demequina, Eubacterium, Herbinix, Insoliti spirillum, Lachnospiraceae, Leptotrichia, Listeria, Paludibacter, Porphyromonadaceae, Pseudobutyrivibrio, Rhodobacter, or Thalassospira.
  • the Cas13a protein is from a species of Leptotrichia shahii, Listeria seeligeri, Lachnospiraceae bacterium (such as Lb MA2020, Lb NK4A179, Lb NK4A144) , Clostridium aminophilum (such as Ca DSM 10710) , Carnobacterium gallinarum (such as Cg DSM 4847) , Paludibacterpropionicigenes (such as Pp WB4) , Listeria weihenstephanensis (such as Lw FSL R9-0317) , Listeriaceae bacterium (such as Lb FSL M6-0635) , Leptotrichia wadei (such as Lw F0279) , Rhodobacter capsulatus (such as Rc SB 1003, Rc R121, Rc DE442) , Leptotrichia buccalis (such as Lb C-1013-b) , Herbinix hemicellulos
  • the Cas13a is any one of Cas13a disclosed in WO2020/028555 (incorporated herein by reference) .
  • the Cas13 protein is a Cas13b protein.
  • the Cas13b protein is from a species of the genus Alistipes, Bacteroides, Bacteroidetes, Bergeyella, Capnocytophaga, Chryseobacterium, Flavobacterium, Myroides, Paludibacter, Phaeodactylibacter, Porphyromonas, Prevotella, Psychroflexus, Reichenbachiella, Riemerella, or Sinomicrobium.
  • the Cas13b protein is from a species Alistipes sp.
  • Bacteroides pyogenes such as Bp F0041
  • Bacteroidetes bacterium such as Bb GWA2319
  • Bergeyella zoohelcum such as Bz ATCC 43767
  • Capnocytophaga canimorsus Capnocytophaga cynodegmi
  • Chryseobacterium carnipullorum Chryseobacterium jejuense
  • Chryseobacterium ureilyticum Flavobacterium branchiophilum
  • Flavobacterium columnare Flavobacterium sp.
  • Myroides odoratimimus (such as Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837) , Paludibacter propionicigenes, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis (such as Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087, Porphyromonas gulae, Porphyromonas sp.
  • COT-052 OH4946 Prevotella aurantiaca, Prevotella buccae (such as Pb ATCC 33574) , Prevotella falsenii, Prevotella intermedia (such as Pi 17, Pi ZT) , Prevotella pallens (such as Pp ATCC 700821 ) , Prevotella pleuritidis, Prevotella saccharolytica (such as Ps F0055) , Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. P5-125, Prevotella sp. P5-60, Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, or Sinomicrobium oceani.
  • the Cas13b is any one of Cas13b disclosed in WO2020/028555 (incorporated herein by reference) .
  • the Cas13 protein is a Cas13c protein. In some embodiments, the Cas13c protein is from a species of the genus Fusobacterium or Anaerosalibacter. In certain embodiments, the Cas13c protein is from a species of Fusobacterium necrophorum (such as Fn subsp. funduliforme ATCC 51357, Fn DJ-2, Fn BFTR-1, Fn subsp. Funduliforme) , Fusobacterium perfoetens (such as Fp ATCC 29250) , Fusobacterium ulcerans (such as Fu ATCC 49185) , or Anaerosalibacter sp. ND1.
  • Fusobacterium necrophorum such as Fn subsp. funduliforme ATCC 51357, Fn DJ-2, Fn BFTR-1, Fn subsp. Funduliforme
  • Fusobacterium perfoetens such as Fp ATCC 29250
  • the Cas13c is any one of Cas13c disclosed in WO2020/028555 (incorporated herein by reference) .
  • the Cas13 protein is a Cas13d protein.
  • the Cas13d protein is from a species of the genus Eubacterium or Ruminococcus.
  • the Cas13d protein is from a species of Eubacterium siraeum, Ruminococcus flavefaciens (such as Rfx XPD3002) , or Ruminococcus albus.
  • Cas13d is CasRx.
  • Cas13d has the amino acid sequence of SEQ ID NO: Cas13d.
  • the Cas13d is any one of Cas13d disclosed in WO2020/028555 (incorporated herein by reference) .
  • the Cas13 protein is a Cas13e protein. . In some embodiments, the Cas13e protein is from a species of the genus Planctomycetes. In certain embodiments, the Cas13e protein has an amino acid sequence of SEQ ID NO: 4, 50 or 51.
  • the direct repeat (DR) sequences for the Cas13e of SEQ ID NOs: 50 and 51 are SEQ ID NOs: 57 and 58, respectively.
  • the Cas13 protein is a Cas13f protein.
  • the Cas13f protein has an amino acid sequence of any one of SEQ ID NOs: 52-56.
  • the direct repeat (DR) sequences for the Cas13f of SEQ ID NOs: 52-56 are SEQ ID NOs: 59-63, respectively.
  • direct repeat sequence may refer to the DNA coding sequence in the CRISPR locus, or to the RNA encoded by the same in crRNA.
  • each T is understood to represent a U.
  • the wild-type Cas effector proteins of the invention can be: (i) any one of SEQ ID NOs: 50-56, such as SEQ ID NO: 50; (ii) an ortholog, paralog, homolog of any one of SEQ ID NOs: 50-56; or (iii) a Class 2 type VI effector enzyme having amino acid sequence identity of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%compared to any one of SEQ ID NOs: 50-56.
  • the Cas13e and Cas13f effector proteins, orthologs, homologs, derivatives and functional fragments thereof are naturally existing. In certain other embodiments, the Cas13e and Cas13f effector proteins, orthologs, homologs, derivatives and functional fragments thereof are not naturally existing, e.g., having at least one amino acid difference compared to a naturally existing sequence.
  • the region spatially close to the endonuclease catalytic domain of the corresponding wild-type Cas13 effector enzyme includes residues within 120, 110, 100, 90, or 80 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXXH domain) in the primary sequence of the Cas13.
  • the region includes residues within 130, 125, 120, 110, 100, 90, or 80 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXXH domain) in the primary sequence of the Cas13e, and residues within 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXH domain) in the primary sequence of the Cas13d.
  • the endonuclease catalytic domain e.g., an RXXXXH domain
  • the region spatially close to the endonuclease catalytic domain of the corresponding wild-type Cas13 effector enzyme includes residues more than 100, 110, 120, or 130 residues away from any residues of the endonuclease catalytic domain in the primary sequence of the Cas13, but are spatially within 1-10 or 5 of a residue of the endonuclease catalytic domain.
  • the endonuclease catalytic domain is a HEPN domain, optionally a HEPN domain comprising an RXXXXH motif.
  • the RXXXXH motif comprises a R ⁇ N/H/K/Q/R ⁇ X 1 X 2 X 3 H sequence.
  • X 1 is R, S, D, E, Q, N, G, or Y
  • X 2 is I, S, T, V, or L
  • X3 is L, F, N, Y, V, I, S, D, E, or A.
  • the RXXXXH motif is an N-terminal RXXXH motif comprising an RNXXXH sequence, such as an RN ⁇ Y/F ⁇ ⁇ F/Y ⁇ SH sequence.
  • the N-terminal RXXXXH motif has a RNYFSH sequence.
  • the N-terminal RXXXXH motif has a RNFYSH sequence.
  • the RXXXXH motif is a C-terminal RXXXXH motif comprising an R ⁇ N/A/R ⁇ ⁇ A/K/S/F ⁇ ⁇ A/L/F ⁇ ⁇ F/H/L ⁇ H sequence.
  • the C-terminal RXXXXH motif may have a RN (A/K) ALH sequence, or a RAFFHH or RRAFFH sequence.
  • region comprises, consists essentially of, or consists of: (a) residues corresponding to residues between residues 1-194, 2-187, 227-242, 620-775, or 634-755 of SEQ ID NO: 4.
  • region comprises, consists essentially of, or consists of residues corresponding to residues between residues 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO: 4; or, (ii) residues corresponding to the HEPN1-1 domain (e.g., residues 90-292) , Helical2 domain (e.g., residues 536-690) , and the HEPN2 domain (e.g., residues 690-967) of SEQ ID NO: Cas13d.
  • the mutation comprises, consists essentially of, or consists of substitutions, within a stretch of 15-20 consecutive amino acids within the region, one or more charged or polar residues to a charge neutral short chain aliphatic residue (such as A) .
  • the stretch is about 16 or 17 residues.
  • the mutation comprises, consists essentially of, or consists of substitutions, within a stretch of 15-20 consecutive amino acids within the region, (a) one or more charged, nitrogen-containing side chain group, bulky (such as F or Y) , aliphatic, and/or polar residues to a charge-neutral short chain aliphatic residue (such as A, V, or I) ; (b) one or more I/L to A substitution (s) ; and/or (c) one or more A to V substitution (s) .
  • a total of about 7, 8, 9, or 10 charged and polar residues within the stretch are substituted.
  • the N-and C-terminal 2 residues of the stretch are substituted to amino acids the coding sequences of which contain a restriction enzyme recognition sequence.
  • the N-terminal two residues may be VF
  • the C-terminal 2 residues may be ED
  • the restriction enzyme is BpiI.
  • Other suitable RE sites are readily envisioned.
  • the RE sites for the N-and C-terminal ends can be, but need not be identical.
  • the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, and T residues. In certain embodiments, the one or more charged or polar residues comprise R, K, H, N, Y, and/or Q residues.
  • one or more Y residue (s) within said stretch is substituted.
  • said one or more Y residues (s) correspond to Y672, Y676, and/or Y715 of wild-type Cas13e. 1 (SEQ ID NO: 4) .
  • said stretch is residues 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO: 4.
  • the mutation leads to reduction or elimination of guide sequence-independent collateral RNase activity.
  • the mutation comprises charge-neutral short chain aliphatic residue substitution (s) corresponding to any one or more of SEQ ID NOs: 37-39, 45, and 48.
  • the mutation leads to enhanced guide sequence-independent collateral RNase activity compared to the wild-type Cas13.
  • the mutation comprises charge-neutral short chain aliphatic residue substitution (s) corresponding to any one or more of SEQ ID NOs: 40-42.
  • the charge-neutral short chain aliphatic residue is A, I, L, V, or G.
  • the charge-neutral short chain aliphatic residue is Ala (A) .
  • the mutation comprises, consists essentially of, or consists of substitutions within 2, 3, 4, or 5 said stretches of 15-20 consecutive amino acids within the region.
  • the mutation comprises, consists essentially of, or consists of: (a) substitutions within 1, 2, 3, 4, or 5 of said stretches of 15-20 consecutive amino acids within the region; (b) a mutation corresponds to a Cas13d mutation of Example 4 that retains at least about 75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d) , and exhibits less than about 27.5%collateral effect of wild-type Cas13d (such as SEQ ID NO: Cas13d) ; (c) a mutation corresponds to the N1V7, N2V7, N2V8 (cfCas13d) , N3V7, or N15V4 mutation of Cas13d mutation; (d) a mutation corresponds to a Cas13d mutation of Example 4 that retains between about 25-75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d) , and exhibit
  • the engineered Cas13 preserves at least about 50%, 60%, 70%, 72.5%, 75%, 80%, 85%, 87.5%, 90%, 95%, 96%, 97%, 97.5%, 98%, or 99%of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA.
  • the engineered Cas13 lacks at least about 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or 100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.
  • the engineered Cas13 preserves at least about 80-90%of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA, and lacks at least about 95-100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.
  • the engineered Cas13 of the invention has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.86%identical to any one of SEQ ID NOs: 6-10, and Cas13d, excluding any one or more of the regions defined by SEQ ID NOs: 16, 20, 24, 28, and 32, and any of the mutation regions in Example 4 or 5.
  • the engineered Cas13 of the invention may differ from the engineered Cas13 of any one of SEQ ID NOs: 6-10 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues, provided that such additional changes do not substantially negatively affect the guide sequence-specific endonuclease activity, and/or do not increase the guide sequence-independent collateral effect.
  • the amino acid sequence contains up to 1, 2, 3, 4, or 5 differences in each of one or more regions defined by SEQ ID NO: 16, 20, 24, 28, and 32, as compared to SEQ ID NOs: 17, 21, 25, 29, and 33, respectively.
  • additional changes in SEQ ID NOs: 17, 21, 25, 29, and/or 33 are possible without substantially negatively affect the guide sequence-specific endonuclease activity, and/or do not increase the guide sequence-independent collateral effect.
  • the engineered Cas13 of the invention has the amino acid sequence of any one of SEQ ID NOs: 6-10. In certain embodiments, the engineered Cas13 of the invention has the amino acid sequence of SEQ ID NO: 9 or 10.
  • the engineered Cas13 of the invention further comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) .
  • the engineered Cas13 may comprise an N-and/or a C-terminal NLS.
  • the invention provides additional derivatives of the subject engineered Cas13, such as those either substantially lacking or having enhanced collateral endonuclease activity, such as Cas13e and Cas13f effector proteins based on any one of SEQ ID NOs: 50-56 (e.g., SEQ ID NOs: 6-10) , or the above orthologs, homologs, derivatives and functional fragments thereof, which comprises another covalently or non-covalently linked protein or polypeptide or other molecules (such as detection reagents or drug /chemical moieties) .
  • SEQ ID NOs: 50-56 e.g., SEQ ID NOs: 6-10
  • orthologs, homologs, derivatives and functional fragments thereof which comprises another covalently or non-covalently linked protein or polypeptide or other molecules (such as detection reagents or drug /chemical moieties) .
  • Such other proteins /polypeptides /other molecules can be linked through, for example, chemical coupling, gene fusion, or other non-covalent linkage (such as biotin-streptavidin binding) .
  • Such derived proteins do not affect the function of the original protein, such as the ability to bind a guide RNA /crRNA of the invention (described herein below) to form a complex, the RNase activity, and the ability to bind to and cleave a target RNA at a specific site, under the guidance of the crRNA that is at least partially complementary to the target RNA.
  • such derived proteins do retain the characteristics of the subject engineered Cas13 either lacking or having enhanced collateral endonuclease activity.
  • the engineered Cas13 upon binding of the RNP complex of the subject engineered Cas13 (or derivative thereof) to the target RNA, the engineered Cas13 either does not exhibit substantial (or detectable) or has enhanced collateral RNase activity.
  • Such derivation may be used, for example, to add a nuclear localization signal (NLS, such as SV40 large T antigen NLS) to enhance the ability of the subject Cas13, e.g., Cas13e and Cas13f effector proteins, to enter cell nucleus.
  • NLS nuclear localization signal
  • Such derivation can also be used to add a targeting molecule or moiety to direct the subject Cas13, e.g., Cas13e and Cas13f effector proteins, to specific cellular or subcellular locations.
  • Such derivation can also be used to add a detectable label to facilitate the detection, monitoring, or purification of the subject Cas13, e.g., Cas13e and Cas13f effector proteins.
  • Such derivation can further be used to add a deamination enzyme moiety (such as one with adenine or cytosine deamination activity) to facilitate RNA base editing.
  • the derivation can be through adding any of the additional moieties at the N-or C-terminal of the subject Cas13 effector proteins, or internally (e.g., internal fusion or linkage through side chains of internal amino acids) .
  • the invention provides conjugates of the subject engineered Cas13, such as those either substantially lacking or having enhanced substantially lacking collateral endonuclease activity, such as Cas13e and Cas13 f effector proteins based on any one of SEQ ID NOs: 50-56 (e.g., SEQ ID NOs: 6-10) , or the above orthologs, homologs, derivatives and functional fragments thereof, which are conjugated with moieties such as other proteins or polypeptides, detectable labels, or combinations thereof.
  • SEQ ID NOs: 50-56 e.g., SEQ ID NOs: 6-10
  • moieties such as other proteins or polypeptides, detectable labels, or combinations thereof.
  • conjugated moieties may include, without limitation, localization signals, reporter genes (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP) , labels (e.g., fluorescent dye such as FITC, or DAPI) , NLS, targeting moieties, DNA binding domains (e.g., MBP, Lex A DBD, Gal4 DBD) , epitope tags (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc) , transcription activation domains (e.g., VP64 or VPR) , transcription inhibition domains (e.g., KRAB moiety or SID moiety) , nucleases (e.g., FokI) , deamination domain (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD) , methylase, demethylase, transcription release factor, HDAC,
  • the conjugate may include one or more NLSs, which can be located at or near N-terminal, C-terminal, internally, or combination thereof.
  • the linkage can be through amino acids (such as D or E, or S or T) , amino acid derivatives (such as Ahx, ⁇ -Ala, GABA or Ava) , or PEG linkage.
  • conjugations do not affect the function of the original engineered protein, such as those either substantially lacking or having enhanced collateral effect, such as the ability to bind a guide RNA /crRNA of the invention (described herein below) to form a complex, and the ability to bind to and cleave a target RNA at a specific site, under the guidance of the crRNA that is at least partially complementary to the target RNA.
  • the invention provides fusions of the subject engineered Cas13, such as those either substantially lacking or having enhanced collateral endonuclease activity, such as Cas13e and Cas13f effector proteins based on any one of SEQ ID NOs: 50-56 (e.g., SEQ ID NOs: 6-10) , or the above orthologs, homologs, derivatives and functional fragments thereof, which fusions are with moieties such as localization signals, reporter genes (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP) , NLS, protein targeting moieties, DNA binding domains (e.g., MBP, Lex A DBD, Gal4 DBD) , epitope tags (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc) , transcription activation domains (e.g., VP64 or VPR) , transcription inhibition domains (e.g.,
  • the fusion may include one or more NLSs, which can be located at or near N-terminal, C-terminal, internally, or combination thereof.
  • conjugations do not affect the function of the original engineered Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, such as the ability to bind a guide RNA /crRNA of the invention (described herein below) to form a complex, the RNase activity, and the ability to bind to and cleave a target RNA at a specific site, under the guidance of the crRNA that is at least partially complementary to the target RNA.
  • the invention provides a polynucleotide encoding the engineered Cas13 of the invention.
  • the polynucleotide may comprise: (i) a polynucleotide encoding any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral effect, e.g., those based on Cas13e or Cas13f effector proteins of SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, functional fragments, fusions thereof; (ii) a polynucleotide of any one of SEQ ID NOs: 11-15; or (iii) a polynucleotide comprising (i) and (ii) .
  • the polynucleotide of the invention is codon-optimized for expression in a eukaryote, a mammal (such as a human or a non-human mammal) , a plant, an insect, a bird, a reptile, a rodent (e.g., mouse, rat) , a fish, a worm /nematode, or a yeast.
  • a mammal such as a human or a non-human mammal
  • a plant an insect, a bird, a reptile, a rodent (e.g., mouse, rat) , a fish, a worm /nematode, or a yeast.
  • the invention provides a polynucleotide having (i) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotides additions, deletions, or substitutions compared to the subject polynucleotide described above; (ii) at least 50%, 60%, 70%, 80%, 90%, 95%, or 97%sequence identity to the subject polynucleotide described above; (iii) hybridize under stringent conditions with the subject polynucleotide described above or any of (i) and (ii) ; or (iv) is a complement of any of (i) - (iii) .
  • one or more e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
  • the invention provides a vector comprising or encompassing any one of the polynucleotides of the invention described herein.
  • the vector can be a cloning vector, or an expression vector.
  • the vector can be a plasmid, phagemid, or cosmid, just to name a few.
  • the vector can be used to express the polynucleotide in a mammalian cell, such as a human cell, any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., the subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, functional fragments, fusions thereof; or any of the polynucleotide of the invention; or any of the complex of the invention.
  • a mammalian cell such as a human cell
  • any one of the engineered Cas13 such as those either substantially lacking or having enhanced collateral activity
  • the subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 such as SEQ ID NOs: 6-10)
  • orthologs, homologs, derivatives, functional fragments, fusions thereof or any of the polynucleotide
  • the polynucleotide is operably linked to a promoter and optionally an enhancer.
  • the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue specific promoter.
  • the vector is a plasmid.
  • thc vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex viral (HSV) vector, an AAV vector, or a lentiviral vector.
  • the AAV vector is a recombinant AAV vector of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV 11, AAV 12, or AAV 13. In certain embodiments.
  • Another aspect of the invention provides a delivery system comprising (1) a delivery vehicle, and (2) the engineered Cas13 of the invention, the polynucleotide of the invention, or the vector of the invention.
  • the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.
  • a further aspect of the invention provides a cell or a progeny thereof, comprising the engineered Cas13 of the invention, the polynucleotide of the invention, or the vector of the invention.
  • the cell can be a prokaryote such as E. coli, or a cell from a eukaryote such as yeast, insect, plant, animal (e.g., mammal including human and mouse) .
  • the cell can be isolated primary cell (such as bone marrow cells for ex vivo therapy) , or established cell lines such as tumor cell lines, 293T cells, or stem cells, iPCs, etc.
  • the cell or progeny thereof is a eukaryotic cell (e.g., a non-human mammalian cell, a human cell, or a plant cell) or a prokaryotic cell (e.g., a bacteria cell) .
  • a eukaryotic cell e.g., a non-human mammalian cell, a human cell, or a plant cell
  • a prokaryotic cell e.g., a bacteria cell
  • a further aspect of the invention provides a non-human multicellular eukaryote comprising the cell of the invention.
  • the non-human multicellular eukaryote is an animal (e.g., rodent or primate) model for a human genetic disorder.
  • the invention provides a complex comprising: (i) a protein composition of any one of the subject engineered Cas13, such as those either substantially lacking or having enhanced collateral endonuclease activity, e.g., engineered Cas13e or Cas13f effector protein, or orthologs, homologs, derivatives, conjugates, functional fragments thereof, conjugates thereof, or fusions thereof; and (ii) a polynucleotide composition, comprising an isolated polynucleotide comprising a cognate DR sequence for said engineered Cas13 effector enzyme, and a spacer /guide sequence complementary to at least a portion of a target RNA.
  • a protein composition of any one of the subject engineered Cas13 such as those either substantially lacking or having enhanced collateral endonuclease activity, e.g., engineered Cas13e or Cas13f effector protein, or orthologs, homologs, derivatives, conjugates, functional fragments thereof, conjugates thereof, or fusions thereof
  • the DR sequence is at the 3’ end of the spacer sequence.
  • the DR sequence is at the 5’ end of the spacer sequence.
  • the polynucleotide composition is the guide RNA /crRNA of the subject engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., engineered Cas13e or Cas13f system, which does not include a tracrRNA.
  • the spacer sequence is at least about 10 nucleotides, or between 10-60, 15-50, 20-50, 25-40, 25-50, or 19-50 nucleotides.
  • the invention provides a eukaryotic cell comprising a subject complex comprising a subject engineered Cas13, said complex comprising: (1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA, and a direct repeat (DR) sequence 5’ or 3’ to the spacer sequence; and, (2) a subject engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, such as a subject engineered Cas13e or Cas13f effetcor enzyme based on a wild-type having an amino acid sequence of any one of SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or a derivative or functional fragment of said Cas; wherein the Cas, the derivative, and the functional fragment of said Cas, are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.
  • a subject engineered Cas13 such as those either substantially lacking or having enhanced collateral activity, such as a subject engineered Cas13e or Cas13f effetcor enzyme
  • the invention provides a composition
  • a composition comprising: (i) a first (protein) composition selected from any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, conjugates, functional fragments, fusions thereof; and (ii) a second (nucleotide) composition comprising an RNA encompassing a guide RNA /crRNA, particularly a spacer sequence, or a coding sequence for the same.
  • a first (protein) composition selected from any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homolog
  • the guide RNA may comprise a DR sequence, and a spacer sequence which can complement or hybridize with a target RNA.
  • the guide RNA can form a complex with the first (protein) composition of (i) .
  • the DR sequence can be the polynucleotide of the invention.
  • the DR sequence can be at the 5-or 3’-end of the guide RNA.
  • the composition (such as (i) and/or (ii) ) is non-naturally occurring or modified from a naturally occurring composition.
  • the target sequence is an RNA from a prokaryote or a eukaryote, such as a non-naturally existing RNA.
  • the target RNA may be present inside a cell, such as in the cytosol or inside an organelle.
  • the protein composition may have an NLS that can be located at its N-or C-terminal, or internally.
  • the invention provides a composition comprising one or more vectors of the invention, said one or more vectors comprise: (i) a first polynucleotide that encodes any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, such as a subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, functional fragments, fusions thereof; optionally operably linked to a first regulatory element; and (ii) a second polynucleotide that encodes a guide RNA of the invention; optionally operably linked to a second regulatory element.
  • a first polynucleotide that encodes any one of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, such as a subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NO
  • the first and the second polynucleotides can be on different vectors, or on the same vector.
  • the guide RNA can form a complex with the protein product encoded by the first polynucleotide, and comprises a DR sequence (such as any one of the 4th aspect) and a spacer sequence that can bind to /complement with a target RNA.
  • the first regulatory element is a promoter, such as an inducible promoter.
  • the second regulatory element is a promoter, such as an inducible promoter.
  • the target sequence is an RNA from a prokaryote or a eukaryote, such as a non-naturally existing RNA.
  • the target RNA may be present inside a cell, such as in the cytosol or inside an organelle.
  • the protein composition may have an NLS that can be located at its N-or C-terminal, or internally.
  • the vector is a plasmid.
  • the vector is a viral vector based on a retrovirus, a replication incompetent retrovirus, adenovirus, replication incompetent adenovirus, or AAV.
  • the vector can self-replicate in a host cell (e.g., having a bacterial replication origin sequence) .
  • the vector can integrate into a host genome and be replicated therewith.
  • the vector is a cloning vector.
  • the vector is an expression vector.
  • the invention further provides a delivery composition for delivering any of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., a subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, conjugates, functional fragments, fusions thereof of the invention; the polynucleotide of the invention; the complex of the invention; the vector of the invention; the cell of the invention, and the composition of the invention.
  • a subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 such as SEQ ID NOs: 6-10)
  • orthologs, homologs, derivatives, conjugates, functional fragments, fusions thereof of the invention the polynucleotide of the invention
  • the complex of the invention the vector of the invention
  • the cell of the invention the composition of the invention.
  • the delivery can be through any one known in the art, such as transfection, lipofection, electroporation, gene gun, microinjection, sonication, calcium phosphate transfection, cation transfection, viral vector delivery, etc., using vehicles such as liposome (s) , nanoparticle (s) , exosome (s) , microvesicle (s) , a gene-gun or one or more viral vector (s) .
  • vehicles such as liposome (s) , nanoparticle (s) , exosome (s) , microvesicle (s) , a gene-gun or one or more viral vector (s) .
  • the invention further provides a kit comprising any one or more of the following: any of the engineered Cas13, such as those either substantially lacking or having enhanced collateral activity, e.g., a subject engineered Cas13e or Cas13f effector proteins based on SEQ ID NOs: 50-56 (such as SEQ ID NOs: 6-10) , or orthologs, homologs, derivatives, conjugates, functional fragments, fusions thereof of the invention; the polynucleotide of the invention; the complex of the invention; the vector of the invention; the cell of the invention, and the composition of the invention.
  • the kit may further comprise an instruction for how to use the kit components, and/or how to obtain additional components from 3 rd party for use with the kit components. Any component of the kit can be stored in any suitable container.
  • One aspect of the invention provides engineered Cas13, such as those either substantially lacking or having enhanced collateral activity.
  • the Cas13 effector enzyme is a Class 2, type VI effector enzyme having two strictly conserved RX4-6H (RXXXXH) -like motifs, characteristic of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains.
  • the CRISPR Class 2, type VI effectors that contain two HEPN domains have been previously characterized and include, for example, CRISPR Cas13a (C2c2) , Cas13b, Cas13c, Cas13d (including the engineered variant CasRx) , Cas13c, and Cas13f.
  • HEPN domains have been shown to be RNase domains and confer the ability to bind to and cleave target RNA molecule.
  • the target RNA may be any suitable form of RNA, including but not limited to mRNA, tRNA, ribosomal RNA, non-coding RNA, IncRNA (long non-coding RNA) , and nuclear RNA.
  • the engineered Cas13 proteins recognize and cleave RNA targets located on the coding strand of open reading frames (ORFs) .
  • the Class 2 type VI Cas13 effector enzyme is of the subtype Type VI-E and VI-F, or Cas13e or Cas13f (such as SEQ ID NOs: 50-56) .
  • Type VI-E and VI-F CRISPR-Cas effector proteins are significantly smaller (e.g., about 20%fewer amino acids) than even the smallest previously identified Type VI-D/Casl3d effectors (see FIG. 15) , and have less than 30%sequence similarity in one to one sequence alignments to other previously described effector proteins, including the phylogenetically closest relatives Cas13b.
  • Class 2, subtypes VI-E and VI-F effectors can be used in a variety of applications, and are particularly suitable for therapeutic applications since they are significantly smaller than other effectors (e.g., CRISPR Cas13a, Cas13b, Cas13c, and Cas13d/CasRx effectors) which allows for the packaging of the nucleic acids encoding the effectors and their guide RNA coding sequences into delivery systems having size limitations, such as the AAV vectors.
  • CRISPR Cas13a, Cas13b, Cas13c, and Cas13d/CasRx effectors e.g., CRISPR Cas13a, Cas13b, Cas13c, and Cas13d/CasRx effectors
  • Exemplary Type VI-D CRISPR-Cas effector proteins include Cas13d, such as SEQ ID NO: Cas13d.
  • Exemplary Type VI-E and VI-F CRISPR-Cas effector proteins are provided in the table below.
  • the two RX4-6H (RXXXXH) motifs in each effector are double-underlined.
  • the C-terminal motif may have two possibilities due to the RR and HH sequences flanking the motif. Mutations at one or both such domains may create an RNase dead version (or “dCas) of the Cas13e and Cas13f effector proteins, homologs, orthologs, fusions, conjugates, derivatives, or functional fragments thereof, while substantially maintaining their ability to bind the guide RNA and the target RNA complementary to the guide RNA.
  • dCas RNase dead version
  • a subject engineered Cas13 effector enzyme such as those either substantially lacking or having enhanced collateral activity is based on a “derivative” of a wild-type Type VI-D, Type VI-E and VI-F CRISPR-Cas effector proteins, said derivative having an amino acid sequence with at least about 80%sequence identity to the amino acid sequence of any one of SEQ ID NOs: Cas13d, and 50-56 above (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) .
  • Such derivative Cas effectors sharing significant protein sequence identity to any one of SEQ ID NOs: Cas13d, and 50-56 have retained at least one of the functions of the Cas of SEQ ID NOs: Cas13d, and 50-56 (see below) , such as the ability to bind to and form a complex with a crRNA comprising at least one of the DR sequences of Cas13d, and SEQ ID NOs: 57-63.
  • a Cas13e For example, a Cas13e.
  • 1 derivative may share 85%amino acid sequence identity to SEQ ID NO: 50, 51, 52, 53, 54, 55, or 56, respectively, and retains the ability to bind to and form a complex with a crRNA having a DR sequence of SEQ ID NO: 57, 58, 59, 60, 61, 62, or 63, respectively.
  • sequence identity between the derivative and the wild-type Cas13 is based on regions outside the regions defined by the mutant regions in Examples 1, 2, 4 and 5, such as SEQ ID NOs: 16, 20, 24, 28, and 32.
  • the derivative comprises conserved amino acid residue substitutions. In some embodiments, the derivative comprises only conserved amino acid residue substitutions (i.e., all amino acid substitutions in the derivative are conserved substitutions, and there is no substitution that is not conserved) .
  • the derivative comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions into any one of the wild-type sequences of Cas13d, and SEQ ID NOs: 50-56.
  • the insertion and/or deletion maybe clustered together, or separated throughout the entire length of the sequences, so long as at least one of the functions of the wild-type sequence is preserved.
  • Such functions may include the ability to bind the guide/crRNA, the RNase activity, the ability to bind to and/or cleave the target RNA complementary to the guide/crRNA.
  • the insertions and/or deletions are not present in the RXXXXH motifs, or within 5, 10, 15, or 20 residues from the RXXXXH motifs.
  • the derivative has retained the ability to bind guide RNA/crRNA.
  • the derivative has retained the guide/crRNA-activated RNase activity.
  • the derivative has retained the ability to bind target RNA and/or cleave the target RNA in the presence of the bound guide/crRNA that is complementary in sequence to at least a portion of the target RNA.
  • the derivative has completely or partially lost the guide/crRNA-activated RNase activity, due to, for example, mutations in one or more catalytic residues of the RNA-guided RNase.
  • Such derivatives are sometimes referred to as dCas, such as dCasl3d and dCas13e. 1.
  • the derivative may be modified to have diminished nuclease/RNase activity, e.g., nuclease inactivation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%as compared with the counterpart wild type proteins.
  • the nuclease activity can be diminished by several methods known in the art, e.g., introducing mutations into the nuclease (catalytic) domains of the proteins.
  • catalytic residues for the nuclease activities are identified, and these amino acid residues can be substituted by different amino acid residues (e.g., glycine or alanine) to diminish the nuclease activity.
  • the amino acid substitution is a conservative amino acid substitution.
  • the amino acid substitution is a non-conservative amino acid substitution.
  • the modification comprises one or more mutations (e.g., amino acid deletions, insertions, or substitutions) in at least one HEPN domain. In some embodiments, there is one, two, three, four, five, six, seven, eight, nine, or more amino acid substitutions in at least one HEPN domain.
  • mutations e.g., amino acid deletions, insertions, or substitutions
  • the one or more mutations comprise a substitution (e.g., an alanine substitution) at an amino acid residue corresponding to R84, H89, R739, H744, R740, H745 of SEQ ID NO: 50 or R97, H102, R770, H775 of SEQ ID NO: 51 or R77, H82, R764, H769 of SEQ ID NO: 52, or R79, H84, R766A, H771 of SEQ ID NO: 53, or R79, H84, R766, H771 of SEQ ID NO: 54, or R89, H94, R773, H778 of SEQ ID NO: 55, or R89, H94, R777, H782 of SEQ ID NO: 56.
  • a substitution e.g., an alanine substitution
  • the one or more mutations comprises, consists essentially of, or consists of: (a) substitutions within 1, 2, 3, 4, or 5 of said stretches of 15-20 consecutive amino acids within the region; (b) a mutation corresponds to a Cas13d mutation of Example 4 that retains at least about 75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d) , and exhibits less than about 27.5%collateral effect of wild-type Cas13d (such as SEQ ID NO: Cas13d) ; (c) a mutation corresponds to the N1V7, N2V7, N2V8 (cfCas13d) , N3V7, or N15V4 mutation of Cas13d mutation; (d) a mutation corresponds to a Cas13d mutation of Example 4 that retains between about 25-75%of guide RNA-specific cleavage of wild-type Cas13d (such as SEQ ID NO: Cas13d)
  • the one or more mutations or the two or more mutations may be in a catalytically active domain of the effector protein comprising a HEPN domain, or a catalytically active domain which is homologous to a HEPN domain.
  • the effector protein comprises one or more of the following mutations: R84A, H89A, R739A, H744A, R740A, H745A (wherein amino acid positions correspond to amino acid positions of Cas13e. 1) .
  • FIGs. 23A-23J provides an exemplary multisequence alignment of several representative Cas13 family enzymes.
  • One of skill in the art can readily map the mutations in any Cas13 family protein sharing substantial sequence homology/identical to any of the sequences in FIGs. 23A-23J and 24A-24M, in order to determine the mutations “corresponding to” the exemplified Cas13d and Cas13e mutations described herein.
  • one or more mutations abolishes catalytic activity of the protein completely or partially (e.g. altered cleavage rate, altered specificity, etc. ) .
  • exemplary (catalytic) residue mutations include: R97A, H102A, R770A, H775A of Cas13e. 2, or R77A, H82A, R764A, H769A of Cas13f. 1, or R79A, H84A, R766A, H771A of Cas13f. 2, or R79A, H84A, R766A, H771A of Cas13f. 3, or R89A, H94A, R773A, H778A of Cas13f. 4, or R89A, H94A, R777A, H782A of Cas13f. 5.
  • any of the R and/or H residues herein may be replaced not be A but by G, V, or I.
  • the presence of at least one of these mutations results in a derivative having reduced or diminished guide sequence-dependent RNase activity as compared to the corresponding wild-type protein lacking the mutations.
  • the additional presence of any one of the mutations in the subject engineered Cas13 substantially lacking collateral effect can reduce/eliminate off-target effect resulting from non-specific RNA binding.
  • the effector protein as described herein is a “dead” effector protein, such as a dead Cas13e or Cas13f effector protein (i.e. dCas13e and dCas13f) .
  • the effector protein has one or more mutations in HEPN domain 1 (N-terminal) .
  • the effector protein has one or more mutations in HEPN domain 2 (C-terminal) .
  • the effector protein has one or more mutations in HEPN domain 1 and HEPN domain 2.
  • the inactivated Cas or derivative or functional fragment thereof can be fused or associated with one or more heterologous/functional domains (e.g., via fusion protein, linker peptides, “GS” linkers, etc. ) .
  • These functional domains can have various activities, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, base-editing activity, and switch activity (e.g., light inducible) .
  • the functional domains are Krüppel associated box (KRAB) , SID (e.g.
  • SID4X SID4X
  • VPR VPR
  • VP16 Fok1, P65, HSF1, MyoD1
  • Adenosine Deaminase Acting on RNA such as ADAR1, ADAR2, APOBEC, cytidine deaminase (AID) , TAD, mini-SOG, APEX, and biotin-APEX.
  • the functional domain is a base editing domain, e.g., ADAR1 (including wild-type or ADAR2DD version thereof, with or without the E1008Q and/or the E488Q mutation (s) ) , ADAR2 (including wild-type or ADAR2DD version thereof, with or without the E1008Q and/or the E488Q mutation (s) ) , APOBEC, or AID.
  • ADAR1 including wild-type or ADAR2DD version thereof, with or without the E1008Q and/or the E488Q mutation (s)
  • ADAR2 including wild-type or ADAR2DD version thereof, with or without the E1008Q and/or the E488Q mutation (s)
  • APOBEC e.g., AID.
  • the functional domain may comprise one or more nuclear localization signal (NLS) domains.
  • the one or more heterologous functional domains may comprise at least two or more NLS domains.
  • the one or more NLS domain (s) may be positioned at or near or in proximity to a terminus of the effector protein (e.g., Cas13e/Cas13f effector proteins) and if two or more NLSs, each of the two may be positioned at or near or in proximity to a terminus of the effector protein (e.g., Cas13e/Cas13f effector proteins) .
  • At least one or more heterologous functional domains may be at or near the amino-terminus of the effector protein and/or wherein at least one or more heterologous functional domains is at or near the carboxy-terminus of the effector protein.
  • the one or more heterologous functional domains may be fused to the effector protein.
  • the one or more heterologous functional domains may be tethered to the effector protein.
  • the one or more heterologous functional domains may be linked to the effector protein by a linker moiety.
  • multiple e.g., two, three, four, five, six, seven, eight, or more
  • identical or different functional domains are present.
  • the functional domain e.g., a base editing domain
  • an RNA-binding domain e.g., MS2
  • the functional domain is associated to or fused via a linker sequence (e.g., a flexible linker sequence or a rigid linker sequence) .
  • a linker sequence e.g., a flexible linker sequence or a rigid linker sequence.
  • Exemplary linker sequences and functional domain sequences are provided in table below.
  • the positioning of the one or more functional domains on the inactivated Cas proteins is one that allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect.
  • the functional domain is a transcription activator (e.g., VP16, VP64, or p65)
  • the transcription activator is placed in a spatial orientation that allows it to affect the transcription of the target.
  • a transcription repressor is positioned to affect the transcription of the target
  • a nuclease e.g., Fokl
  • the functional domain is positioned at the N-terminus of the Cas/dCas.
  • the functional domain is positioned at the C-terminus of the Cas/dCas.
  • the inactivated CRISPR-associated protein (dCas) is modified to comprise a first functional domain at the N-terminus and a second functional domain at the C-terminus.
  • a “functional fragment, ” as used herein, refers to a fragment of a wild-type Cas13 protein such as any one of SEQ ID NOs: Cas13d, and 50-56, or a derivative thereof, that has less-than full-length sequence.
  • the deleted residues in the functional fragment can be at the N-terminus, the C-terminus, and/or internally.
  • the functional fragment retains at least one function of the wild-type VI-D, VI-E or VI-F Cas, or at least one function of its derivative. Thus a functional fragment is defined specifically with respect to the function at issue.
  • a functional fragment wherein the function is the ability to bind crRNA and target RNA, may not be a functional fragment with respect to the RNase function, because losing the RXXXXH motifs at both ends of the Cas may not affect its ability to bind a crRNA and target RNA, but may eliminate/destroy the RNase activity.
  • the engineered Cas13 of the invention including a functional fragment of an engineered Cas13 that substantially retains the corresponding wild-type Cas13’s guide sequence-dependent RNase activity, but substantially lacks collateral activity.
  • the engineered Class 2 type VI effector proteins or derivatives thereof or functional fragments thereof lacks about 30, 60, 90, 120, 150, or about 180 residues from the N-terminus.
  • the engineered Class 2 type VI effector proteins or derivatives thereof or functional fragments thereof lacks about 30, 60, 90, 120, or about 150 residues from the C-terminus.
  • the engineered Class 2 type VI effector proteins or derivatives thereof or functional fragments thereof lacks about 30, 60, 90, 120, 150, or about 180 residues from the N-terminus, and lacks about 30, 60, 90, 120, or about 150 residues from the C-terminus.
  • the engineered Class 2 Type VI Cas13 effector proteins or derivatives thereof or functional fragments thereof have RNase activity, e.g., guide/crRNA-activated specific RNase activity.
  • the engineered Class 2 Type VI Cas13 effector proteins or derivatives thereof or functional fragments thereof have no substantial/detectable collateral RNase activity.
  • the present disclosure also provides a split version of the engineered Class 2 type VI Cas13 effector enzyme described herein (e.g., a Type VI-D, VI-E or VI-F CRISPR-Cas effector protein) .
  • the split version of the engineered Cas13 may be advantageous for delivery.
  • the engineered Cas13 is split into two parts of the enzyme, which together substantially comprise a functioning engineered Class 2 type VI Cas13.
  • the split can be done in a way that the catalytic domain (s) are unaffected.
  • the CRISPR-associated protein may function as a nuclease or may be an inactivated enzyme, which is essentially a RNA-binding protein with very little or no catalytic activity (e.g., due to mutation (s) in its catalytic domains) .
  • Split enzymes are described, e.g., in Wright et al., “Rational design of a split-Cas9 enzyme complex, ” Proc. Nat′l. Acad. Sci. 112 (10) : 2984-2989, 2015, which is incorporated herein by reference in its entirety.
  • the nuclease lobe and ⁇ -helical lobe are expressed as separate polypeptides.
  • the crRNA recruits them into a ternary complex that recapitulates the activity of full-length CRISPR-associated proteins and catalyzes site-specific cleavage.
  • the use of a modified crRNA abrogates split-enzyme activity by preventing dimerization, allowing for the development of an inducible dimerization system.
  • the split CRISPR-associated protein can be fused to a dimerization partner, e.g., by employing rapamycin sensitive dimerization domains. This allows the generation of a chemically inducible CRISPR-associated protein for temporal control of the activity of the protein.
  • the CRISPR-associated protein can thus be rendered chemically inducible by being split into two fragments and rapamycin-sensitive dimerization domains can be used for controlled re-assembly of the protein.
  • the split point is typically designed in silico and cloned into the constructs. During this process, mutations can be introduced to the split CRISPR-associated protein and non-functional domains can be removed.
  • the two parts or fragments of the split CRISPR-associated protein can form a full CRISPR-associated protein, comprising, e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%of the sequence of the wild-type CRISPR-associated protein.
  • the CRISPR-associated proteins described herein can be designed to be self-activating or self-inactivating.
  • the target sequence can be introduced into the coding construct of the CRISPR-associated protein.
  • the CRISPR-associated protein can cleave the target sequence, as well as the construct encoding the protein thereby self-inactivating their expression.
  • Methods of constructing a self-inactivating CRISPR system are described, e.g., in Epstein and Schaffer, Mol. Ther. 24: S50, 2016, which is incorporated herein by reference in its entirety.
  • an additional crRNA expressed under the control of a weak promoter (e.g., 7SK promoter) , can target the nucleic acid sequence encoding the CRISPR-associated protein to prevent and/or block its expression (e.g., by preventing the transcription and/or translation of the nucleic acid) .
  • the transfection of cells with vectors expressing the CRISPR-associated protein, the crRNAs, and crRNAs that target the nucleic acid encoding the CRISPR-associated protein can lead to efficient disruption of the nucleic acid encoding the CRISPR-associated protein and decrease the levels of CRISPR-associated protein, thereby limiting its activity.
  • the activity of the CRISPR-associated protein can be modulated through endogenous RNA signatures (e.g., miRNA) in mammalian cells.
  • a CRISPR-associated protein switch can be made by using a miRNA-complementary sequence in the 5’-UTR of mRNA encoding the CRISPR-associated protein.
  • the switches selectively and efficiently respond to miRNA in the target cells.
  • the switches can differentially control the Cas activity by sensing endogenous miRNA activities within a heterogeneous cell population. Therefore, the switch systems can provide a framework for cell-type selective activity and cell engineering based on intracellular miRNA information (see, e.g., Hirosawa et al., Nucl. Acids Res. 45 (13) : e118, 2017) .
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity (e.g., engineered Type VI-D, VI-E and VI-F CRISPR-Cas effector proteins) can be inducibly expressed, e.g., their expression can be light-induced or chemically-induced. This mechanism allows for activation of the functional domain in the CRISPR-associated proteins.
  • Light inducibility can be achieved by various methods known in the art, e.g., by designing a fusion complex wherein CRY2 PHR/CIBN pairing is used in split CRISPR-associated proteins (see, e.g., Konermann et al., “Optical control of mammalian endogenous transcription and epigenetic states, ” Nature 500: 7463, 2013.
  • Chemical inducibility can be achieved, e.g., by designing a fusion complex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding domain) pairing is used in split CRISPR-associated proteins. Rapamycin is required for forming the fusion complex, thereby activating the CRISPR-associated proteins (see, e.g., Zetsche et al., “A split-Cas9 architecture for inducible genome editing and transcription modulation, ” Nature Biotech. 33: 2: 139-42, 2015) .
  • FKBP/FRB FK506 binding protein/FKBP rapamycin binding domain
  • expression of the engineered Class 2 type VI Cas13 effectors can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system) , hormone inducible gene expression system (e.g., an ecdysone inducible gene expression system) , and an arabinose-inducible gene expression system.
  • inducible promoters e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system)
  • hormone inducible gene expression system e.g., an ecdysone inducible gene expression system
  • arabinose-inducible gene expression system e.g., anose-inducible gene expression system.
  • RNA targeting effector protein When delivered as RNA, expression of the RNA targeting effector protein can be modulated via a riboswitch, which can sense a small molecule like tetracycline (see, e.g., Goldfless et al., “Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction, ” Nucl. Acids Res. 40: 9: e64-e64, 2012) .
  • inducible CRISPR-associated proteins and inducible CRISPR systems are described, e.g., in U.S. Pat. No. 8,871,445, US Publication No. 2016/0208243, and International Publication No. WO 2016/205764, each of which is incorporated herein by reference in its entirety.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Localization Signal (NLS) attached to the N-terminal or C-terminal of the protein.
  • NLS Nuclear Localization Signal
  • 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 hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence PQPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKR
  • the CRISPR-associated protein comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Export Signal (NES) attached the N-terminal or C-terminal of the protein.
  • NES Nuclear Export Signal
  • a C-terminal and/or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity are mutated at one or more amino acid residues to alter one or more functional activities.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity is mutated at one or more amino acid residues to alter its helicase activity.
  • the engineered Class 2 type VI Cas l3 effectors such as those either substantially lacking or having enhanced collateral activity is mutated at one or more amino acid residues to alter its nuclease activity (e.g., endonuclease activity or exonuclease activity) , such as the collateral nuclease activity that is not dependent on guide sequence.
  • nuclease activity e.g., endonuclease activity or exonuclease activity
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity is mutated at one or more amino acid residues to alter its ability to functionally associate with a guide RNA.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein are capable of cleaving a target RNA molecule.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity is mutated at one or more amino acid residues to alter its cleaving activity.
  • the engineered Class 2 type VI Cas13 effectors, such as those either substantially lacking or having enhanced collateral activity may comprise one or more mutations that render the enzyme incapable of cleaving a target nucleic acid.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity is capable of cleaving the strand of the target nucleic acid that is complementary to the strand to which the guide RNA hybridizes.
  • a engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein can be engineered to have a deletion in one or more amino acid residues to reduce the size of the enzyme while retaining one or more desired functional activities (e.g., nuclease activity and the ability to interact functionally with a guide RNA) .
  • the truncated engineered Class 2 type VI Cas13 effectors, such as those either substantially lacking or having enhanced collateral activity can be advantageously used in combination with delivery systems having load limitations.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein can be fused to one or more peptide tags, including a His-tag, GST-tag, a V5-tag, FLAG-tag, HA-tag, VSV-G-tag, Trx-tag, or myc-tag.
  • peptide tags including a His-tag, GST-tag, a V5-tag, FLAG-tag, HA-tag, VSV-G-tag, Trx-tag, or myc-tag.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein can be fused to a detectable moiety such as GST, a fluorescent protein (e.g., GFP, HcRed, DsRed, CFP, YFP, or BFP) , or an enzyme (such as HRP or CAT) .
  • a detectable moiety such as GST, a fluorescent protein (e.g., GFP, HcRed, DsRed, CFP, YFP, or BFP)
  • an enzyme such as HRP or CAT
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein can be fused to MBP, LexA DNA binding domain, or Gal4 DNA-binding domain.
  • the engineered Class 2 type VI Cas13 effectors such as those either substantially lacking or having enhanced collateral activity described herein can be linked to or conjugated with a detectable label such as a fluorescent dye, including FITC and DAPI.
  • a detectable label such as a fluorescent dye, including FITC and DAPI.
  • the linkage between the engineered Class 2 type VI Cas13 effectors, such as those either substantially lacking or having enhanced collateral activity described herein and the other moiety can be at the N-or C-terminal of the CRISPR-associated proteins, and sometimes even internally via covalent chemical bonds.
  • the linkage can be affected by any chemical linkage known in the art, such as peptide linkage, linkage through the side chain of amino acids such as D, E, S, T, or amino acid derivatives (Ahx, ⁇ -Ala, GABA or Ava) , or PEG linkage.
  • the invention also provides nucleic acids encoding the proteins described herein (e.g., an engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity) .
  • the nucleic acid is a synthetic nucleic acid. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule encoding the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, derivative or functional fragment thereof) . In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methyl cytidine, substituted with pseudouridine, or a combination thereof.
  • the nucleic acid e.g., DNA
  • a regulatory element e.g., a promoter
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a cell-specific promoter.
  • the promoter is an organisrn-specific promoter.
  • Suitable promoters are known in the art and include, for example, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, and a ⁇ -actin promoter.
  • a U6 promoter can be used to regulate the expression of a guide RNA molecule described herein.
  • the nucleic acid (s) are present in a vector (e.g., a viral vector or a phage) .
  • the vector can be a cloning vector, or an expression vector.
  • the vectors can be plasmids, phagemids, Cosmids, etc.
  • the vectors may include one or more regulatory elements that allow for the propagation of the vector in a cell of interest (e.g., a bacterial cell or a mammalian cell) .
  • the vector includes a nucleic acid encoding a single component of a CRISPR-associated (Cas) system described herein.
  • the vector includes multiple nucleic acids, each encoding a component of a CRISPR-associated (Cas) system described herein.
  • the present disclosure provides nucleic acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the nucleic acid sequences described herein, i.e., nucleic acid sequences encoding the engineered Class 2 type VI Cas13 protein substantially lacking collateral activity, derivatives, functional fragments, or guide/crRNA, including the DR sequences.
  • the present disclosure also provides nucleic acid sequences encoding amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequences of the subject engineered Class 2 type VI Cas13 protein substantially lacking collateral activity.
  • the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as the sequences described herein. In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from the sequences described herein.
  • the invention provides amino acid sequences having at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as the sequences described herein.
  • the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from the sequences described herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the length of a reference sequence aligned for comparison purposes should be at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • proteins described herein e.g., an engineered Class 2 type VI Cas13 protein substantially lacking collateral activity
  • the nucleic acid molecule encoding the engineered Class 2 type VI Cas13 protein such as those either substantially lacking or having enhanced collateral activity, derivatives or functional fragments thereof are codon-optimized for expression in a host cell or organism.
  • the host cell may include established cell lines (such as 293T cells) or isolated primary cells.
  • the nucleic acid can be codon optimized for use in any organism of interest, in particular human cells or bacteria.
  • the nucleic acid can be codon-optimized for any prokaryotes (such as E.
  • 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 et al., Nucl. Acids Res. 28: 292, 2000 (incorporated herein by reference in its entirety) . 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. ) .
  • a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans) , or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667) . Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http: //www. kazusa. orjp/codon/and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28: 292 (2000) .
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA) , are also available.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a Cas correspond to the most frequently used codon for a particular amino acid.
  • the CRISPR systems described herein include at least RNA guide (e.g., a gRNA or a crRNA) .
  • RNA guides The architecture of multiple RNA guides is known in the art (see, e.g., International Publication Nos. WO 2014/093622 and WO 2015/070083, the entire contents of each of which are incorporated herein by reference) .
  • the CRISPR systems described herein include multiple RNA guides (e.g., one, two, three, four, five, six, seven, eight, or more RNA guides) .
  • the RNA guide includes a crRNA. In some embodiments, the RNA guide includes a crRNA but not a tracrRNA.
  • the crRNA includes a direct repeat (DR) sequence and a spacer sequence.
  • the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence, preferably at the 3’-end of the spacer sequence.
  • an engineered Class 2 type VI Cas13 protein such as those either substantially lacking or having enhanced collateral activity forms a complex with the mature crRNA, which spacer sequence directs the complex to a sequence-specific binding with the target RNA that is complementary to the spacer sequence, and/or hybridizes to the spacer sequence.
  • the resulting complex comprises the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity and the mature crRNA bound to the target RNA.
  • the direct repeat sequences for the Cas13 systems are generally well conserved, especially at the ends, with, for example, a GCTG for Cas13e and GCTGT for Cas13f at the 5’-end, reverse complementary to a CAGC for Cas13e and ACAGC for Cas13f at the 3’ end.
  • This conservation suggests strong base pairing for an RNA stem-loop structure that potentially interacts with the protein (s) in the locus.
  • the direct repeat sequence when in RNA, comprises the general secondary structure of 5’-S1a-Ba-S2a-L-S2b-Bb-S1b-3’, wherein segments S1a and S1b are reverse complement sequences and form a first stem (S1) having 4 nucleotides in Cas13e and 5 nucleotides in Cas13f; segments Ba and Bb do not base pair with each other and form a symmetrical or nearly symmetrical bulge (B) , and have 5 nucleotides each in Cas13e, and 5 (Ba) and 4 (Bb) or 6 (Ba) and 5 (Bb) nucleotides respectively in Cas13f; segments S2a and S2b are reverse complement sequences and form a second stem (S2) having 5 base pairs in Cas13e and either 6 or 5 base pairs in Cas13f; and L is an 8-nucleotide loop in Cas13e and a 5-nucleotide loop in
  • S1a has a sequence of GCUG in Cas13e and GCUGU in Cas13f.
  • S2a has a sequence of GCCCC in Cas13e and A/G CCUC G/A in Cas13f (wherein the first A or G may be absent) .
  • the direct repeat sequence comprises or consists of a nucleic acid sequence of SEQ ID NOs: 57-63.
  • direct repeat sequence may refer to the DNA coding sequence in the CRISPR locus, or to the RNA encoded by the same in crRNA.
  • each T is understood to represent a U.
  • the direct repeat sequence comprises or consists of a nucleic acid sequence having up to 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides of deletion, insertion, or substitution of SEQ ID NOs: 57-63. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97%of sequence identity with SEQ ID NOs: 57-63 (e.g., due to deletion, insertion, or substitution ofnucleotides in SEQ ID NOs: 57-63) .
  • the direct repeat sequence comprises or consists of a nucleic acid sequence that is not identical to any one of SEQ ID NOs: 57-63, but can hybridize with a complement of any one of SEQ ID NOs: 57-63 under stringent hybridization conditions, or can bind to a complement of any one of SEQ ID NOs: 57-63 under physiological conditions.
  • the deletion, insertion, or substitution does not change the overall secondary structure of that of SEQ ID NOs: 57-63 (e.g., the relative locations and/or sizes of the stems and bulges and loop do not significantly deviate from that of the original stems, bulges, and loop) .
  • the deletion, insert, or substitution may be in the bulge or loop region so that the overall symmetry of the bulge remains largely the same.
  • the deletion, insertion, or substitution may be in the stems so that the length of the stems do not significantly deviate from that of the original stems (e.g., adding or deleting one base pair in each of the two stems correspond to 4 total base changes) .
  • the deletion, insertion, or substitution results in a derivative DR sequence that may have ⁇ 1 or 2 base pair (s) in one or both stems, have ⁇ 1, 2, or 3 bases in either or both of the single strands in the bulge, and/or have ⁇ 1, 2, 3, or 4 bases in the loop region.
  • any of the above direct repeat sequences that is different from any one of SEQ ID NOs: 57-63 retains the ability to function as a direct repeat sequence in the Cas13e or Cas13f proteins, as the DR sequence of SEQ ID NOs: 57-63.
  • the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence of any one of SEQ ID NOs: 57-63, with a truncation of the initial three, four, five, six, seven, or eight 3’ nucleotides.
  • the degree of complementarity between a guide sequence e.g., a crRNA
  • its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of complementarity is 90-100%.
  • the guide RNAs can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200 or more nucleotides in length.
  • the spacer can be between 10-60 nucleotides, 20-50 nucleotides, 25-45 nucleotides, 25-35 nucleotides, or about 27, 28, 29, 30, 31, 32, or 33 nucleotides.
  • the spacer can be between 10-200 nucleotides, 20-150 nucleotides, 25-100 nucleotides, 25-85 nucleotides, 35-75 nucleotides, 45-60 nucleotides, or about 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 nucleotides.
  • mutations can be introduced to the CRISPR systems so that the CRISPR systems can distinguish between target and off-target sequences that have greater than 80%, 85%, 90%, or 95%complementarity.
  • the degree of complementarity is from 80%to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches) .
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity is 100%.
  • cleavage efficiency can be exploited by introduction of mismatches, e.g., one or more mismatches, such as 1 or 2 mismatches between spacer sequence and target sequence, including the position of the mismatch along the spacer/target.
  • mismatches e.g., one or more mismatches, such as 1 or 2 mismatches between spacer sequence and target sequence, including the position of the mismatch along the spacer/target.
  • cleavage efficiency can be modulated. For example, if less than 100%cleavage of targets is desired (e.g., in a cell population) , 1 or 2 mismatches between spacer and target sequence can be introduced in the spacer sequences.
  • Type VI CRISPR-Cas effectors have been demonstrated to employ more than one RNA guide, thus enabling the ability of these effectors, and systems and complexes that include them, to target multiple nucleic acids.
  • the CRISPR systems comprising the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, as described herein include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more) RNA guides.
  • the CRISPR systems described herein include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem.
  • the single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof.
  • the processing capability of the Type VI-E and VI-F CRISPR-Cas effector proteins described herein enables these effectors to be able to target multiple target nucleic acids (e.g., target RNAs) without a loss of activity.
  • the Type VI-E and VI-F CRISPR-Cas effector proteins may be delivered in complex with multiple RNA guides directed to different target RNA.
  • the engineered Class 2 type VI Cas13 protein such as those either substantially lacking or having enhanced collateral activity may be co-delivered with multiple RNA guides, each specific for a different target nucleic acid.
  • the spacer length of crRNAs can range from about 10-50 nucleotides, such as 15-50 nucleotides, 20-50 nucleotides, 25-50 nucleotide, or 19-50 nucleotides.
  • the spacer length of a guide RNA is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides.
  • the spacer length is from 15 to 17 nucleotides (e.g., 15, 16, or 17 nucleotides) , 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, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides) , from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides) , or longer.
  • the direct repeat length of the guide RNA is 15-36 nucleotides, is at least 16 nucleotides, is from 16 to 20 nucleotides (e.g., 16, 17, 18, 19, or 20 nucleotides) , is from 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) , is from 30-40 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides) , or is about 36 nucleotides (e.g., 33, 34, 35, 36, 37, 38, or 39 nucleotides) . In some embodiments, the direct repeat length of the guide RNA is 36 nucleotides.
  • the overall length of the crRNA /guide RNA is about 36 nucleotides longer than any one of the spacer sequence length described herein above.
  • the overall length of the crRNA /guide RNA may be between 45-86 nucleotides, or 60-86 nucleotides, 62-86 nucleotides, or 63-86 nucleotides.
  • the crRNA sequences can be modified in a manner that allows for formation of a complex between the crRNA and the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, and successful binding to the target, while at the same time not allowing for successful nuclease activity (i.e., without nuclease activity/without causing indels) .
  • These modified guide sequences are referred to as “dead crRNAs, ” “dead guides, ” or “dead guide sequences. ”
  • These dead guides or dead guide sequences may be catalytically inactive or conformationally inactive with regard to nuclease activity. Dead guide sequences are typically shorter than respective guide sequences that result in active RNA cleavage.
  • dead guides are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective guide RNAs that have nuclease activity.
  • Dead guide sequences of guide RNAs can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length) , from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length) .
  • the disclosure provides non-naturally occurring or engineered CRISPR systems including a functional engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity as described herein, and a crRNA, wherein the crRNA comprises a dead crRNA sequence whereby the crRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a target RNA of interest in a cell without detectable nuclease activity (e.g., RNase activity) .
  • a functional engineered Class 2 type VI Cas13 protein such as those either substantially lacking or having enhanced collateral activity as described herein
  • a crRNA wherein the crRNA comprises a dead crRNA sequence whereby the crRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a target RNA of interest in a cell without detectable nuclease activity (e.g., RNase activity) .
  • dead guides A detailed description of dead guides is described, e.g., in International Publication No. WO 2016/094872, which is incorporated herein by reference in its entirety.
  • Guide RNAs can be generated as components of inducible systems.
  • the inducible nature of the systems allows for spatio-temporal control of gene editing or gene expression.
  • the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.
  • the transcription of guide RNA can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems) , hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems) , and arabinose-inducible gene expression systems.
  • inducible promoters e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems)
  • hormone inducible gene expression systems e.g., ecdysone inducible gene expression systems
  • arabinose-inducible gene expression systems e.g., ecdysone inducible gene expression systems
  • inducible systems include, e.g., small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.
  • RNA is amenable to both 5’ and 3’ end conjugations with a variety of functional moieties including fluorescent dyes, polyethylene glycol, or proteins.
  • modifying an oligonucleotide with a 2’-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing.
  • a 2’-OMe modification can affect how the oligonucleotide interacts with transfection reagents, proteins or any other molecules in the cell. The effects of these modifications can be determined by empirical testing.
  • the crRNA includes one or more phosphorothioate modifications. In some embodiments, the crRNA includes one or more locked nucleic acids for the purpose of enhancing base pairing and/or increasing nuclease resistance.
  • RNA guides e.g., crRNAs
  • the optimized length of an RNA guide can be determined by identifying the processed form of crRNA (i.e., a mature crRNA) , or by empirical length studies for crRNA tetraloops.
  • the crRNAs can also include one or more aptamer sequences.
  • Aptamers are oligonucleotide or peptide molecules have a specific three-dimensional structure and can bind to a specific target molecule.
  • the aptamers can be specific to gene effectors, gene activators, or gene repressors.
  • the aptamers can be specific to a protein, which in turn is specific to and recruits and/or binds to specific gene effectors, gene activators, or gene repressors.
  • the effectors, activators, or repressors can be present in the form of fusion proteins.
  • the guide RNA has two or more aptamer sequences that are specific to the same adaptor proteins.
  • the two or more aptamer sequences are specific to different adaptor proteins.
  • the adaptor proteins can include, e.g., MS2, PP7, Q ⁇ , F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ kCb5, ⁇ kCb8r, ⁇ kCb12r, ⁇ kCb23r, 7s, and PRR1.
  • the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein.
  • the aptamer sequence is a MS2 binding loop (5’-ggcccAACAUGAGGAUCACCCAUGUCUGCAGgggcc-3’) . In some embodiments, the aptamer sequence is a QBeta binding loop (5’-ggcccAUGCUGUCUAAGACAGCAUgggcc-3’) . In some embodiments, the aptamer sequence is a PP7 binding loop (5’-ggcccUAAGGGUUUAUAUGGAAACCCUUAgggcc-3’.
  • a dctailed description of aptamers can be found, e.g., in Nowak et al., “Guide RNA engineering for versatile Cas9 functionality, ” Nucl. Acid. Res., 44 (20) : 9555-9564, 2016; and WO 2016205764, which are incorporated herein by reference in their entirety.
  • the methods make use of chemically modified guide RNAs.
  • guide RNA chemical modifications include, without limitation, incorporation of 2’-O-methyl (M) , 2’-O-methyl 3’-phosphorothioate (MS) , or 2’-O-methyl 3’-thioPACE (MSP) at one or more terminal nucleotides.
  • M 2’-O-methyl
  • MS 2’-O-methyl 3’-phosphorothioate
  • MSP 2’-O-methyl 3’-thioPACE
  • Such chemically modified guide RNAs can comprise increased stability and increased activity as compared to unmodified guide RNAs, though on- target vs. off-target specificity is not predictable. See, Hendel, Nat Biotechnol. 33 (9) : 985-9, 2015, incorporated by reference) .
  • Chemically modified guide RNAs may further include, without limitation, RNAs with phosphorothioate linkages and locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2’ and 4’ carbons of the ribose ring.
  • LNA locked nucleic acid
  • the invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest.
  • the nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers.
  • the one or more aptamers may be capable of binding a bacteriophage coat protein.
  • the bacteriophage coat protein may be selected from the group comprising Q ⁇ , F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ Cb5, ⁇ Cb8r, ⁇ Cb12r, ⁇ Cb23r, 7s and PRR1.
  • the bacteriophage coat protein is MS2.
  • the target RNA can be any RNA molecule of interest, including naturally-occurring and engineered RNA molecules.
  • the target RNA can be an mRNA, a tRNA, a ribosomal RNA (rRNA) , a microRNA (miRNA) , an interfering RNA (siRNA) , a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA.
  • the target nucleic acid is associated with a condition or disease (e.g., an infectious disease or a cancer) .
  • a condition or disease e.g., an infectious disease or a cancer
  • the systems described herein can be used to treat a condition or disease by targeting these nucleic acids.
  • the target nucleic acid associated with a condition or disease may be an RNA molecule that is overexpressed in a diseased cell (e.g., a cancer or tumor cell) .
  • the target nucleic acid may also be a toxic RNA and/or a mutated RNA (e.g., an mRNA molecule having a splicing defect or a mutation) .
  • the target nucleic acid may also be an RNA that is specific for a particular microorganism (e.g., a pathogenic bacteria) .
  • One aspect of the invention provides a complex of an engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, such as CRISPR/Cas13e or CRISPR/Cas13f complex, comprising (1) any of the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity (e.g., engineered Cas13e/Cas13f effector proteins, homologs, orthologs, fusions, derivative, conjugates, or functional fragments thereof as described herein) , and (2) any of the guide RNA described herein, each including a spacer sequence designed to be at least partially complementary to a target RNA, and a DR sequence compatible with the engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity (e.g., Cas13d, Cas13e/Cas13f effector proteins) , homologs, orthologs, fusions, derivatives, conjugates, or functional fragments thereof.
  • the complex further comprises the target RNA bound by the guide RNA.
  • the invention also provides a cell comprising any of the complex of the invention.
  • the cell is a prokaryote.
  • the cell is a eukaryote.
  • the CRISPR/Cas systems having the engineered Cas13 e.g., an engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, as described herein, have a wide variety of utilities like the corresponding wild-type Cas13-based systems, including modifying (e.g., deleting, inserting, translocating, inactivating, or activating) a target polynucleotide or nucleic acid in a multiplicity of cell types.
  • the CRISPR systems have a broad spectrum of applications in, e.g., tracking and labeling of nucleic acids, enrichment assays (extracting desired sequence from background) , controlling interfering RNA or miRNA, detecting circulating tumor DNA, preparing next generation library, drug screening, disease diagnosis and prognosis, and treating various genetic disorders.
  • Certain engineered Cas13 effector enzymes have enhanced collateral effect compared to the wild-type, and thus may be better alternatives than the wild-type Cas13 effector enzymes for utilities that take advantage of the enhanced collateral activity, such as DNA/RNA detection (e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK) ) .
  • DNA/RNA detection e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK)
  • SHERLOCK specific high sensitivity enzymatic reporter unlocking
  • the CRISPR systems described herein can be used in RNA detection.
  • wild-type Cas13 such as Cas13e of the invention exhibit non-specific /collateral RNase activity upon activation of its guide RNA-dependent specific RNase activity when the spacer sequence is about 30 nucleotides.
  • the engineered CRISPR-associated proteins of the invention with enhanced collateral activity can be reprogrammed with CRISPR RNAs (crRNAs) to provide a platform for specific RNA sensing.
  • crRNAs CRISPR RNAs
  • activated CRISPR-associated proteins engage in enhanced collateral cleavage of nearby non-targeted RNAs. This crRNA-programmed collateral cleavage activity allows the CRISPR systems to detect the presence of a specific RNA by triggering programmed cell death or by nonspecific degradation of labeled RNA.
  • the SHERLOCK method (Specific High Sensitivity Enzymatic Reporter UnLOCKing) provides an in vitro nucleic acid detection platform with attomolar sensitivity based on nucleic acid amplification and collateral cleavage of a reporter RNA, allowing for real-time detection of the target.
  • the detection can be combined with different isothermal amplification steps.
  • recombinase polymerase amplification RPA
  • T7 transcription to convert amplified DNA to RNA for subsequent detection.
  • SHERLOCK The combination of amplification by RPA, T7 RNA polymerase transcription of amplified DNA to RNA, and detection of target RNA by collateral RNA cleavage-mediated release of reporter signal is referred as SHERLOCK.
  • the invention described herein provides mutant /variant Class 2, Type VI CRISPR/Cas effector enzymes, especially Type VI-D, -E, and -F Cas mutants /variants having enhanced collateral effect, such that they can be more effective in nucleic acid detection assays based on the collateral effect, such as the SHERLOCK assay.
  • Such mutants include any one described in Examples 1, 2, 4, and 5, as well as FIGs. 6, 7, 9-14, 17D, 17E, 19C, and 19D, having at least 80%, 85%, or 87.5%or more collateral cleavage efficiency, and optionally better gRNA-guided cleavage compared to a corresponding wild-type Cas13.
  • such Cas13 mutants have enhanced collateral effect comprises, consists essentially of, or consists of a mutation corresponding to the N2-Y142A, N4-Y193A, N12-Y604A, or N21V7 mutation of Cas13d, or to the M14V2, M16V3, M18V1, M19-G712A, M19-T725A, or M19-C727A mutation of Cas13e.
  • the CRISPR-associated proteins can be used in Northern blot assays, which use electrophoresis to separate RNA samples by size.
  • the CRISPR-associated proteins can be used to specifically bind and detect the target RNA sequence.
  • the CRISPR-associated proteins can also be fused to a fluorescent protein (e.g., GFP) and used to track RNA localization in living cells. More particularly, the CRISPR-associated proteins can be inactivated in that they no longer cleave RNAs as described above.
  • CRISPR-associated proteins can be used to determine the localization of the RNA or specific splice variants, the level of mRNA transcripts, up-or down-regulation of transcripts and disease-specific diagnosis.
  • the CRISPR-associated proteins can be used for visualization of RNA in (living) cells using, for example, fluorescent microscopy or flow cytometry, such as fluorescence-activated cell sorting (FACS) , which allows for high-throughput screening of cells and recovery of living cells following cell sorting.
  • FACS fluorescence-activated cell sorting
  • the CRISPR systems described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH) .
  • MEFISH multiplexed error-robust fluorescence in situ hybridization
  • the CRISPR systems described herein can be used to detect a target RNA in a sample (e.g., a clinical sample, a cell, or a cell lysate) .
  • a sample e.g., a clinical sample, a cell, or a cell lysate
  • the collateral RNase activity of the engineered Cas13 e.g., Type VI-E and/or VI-F CRISPR-Cas effector proteins described herein, is activated when the effector proteins bind to a target nucleic acid when the spacer sequence is of a specific chosen length (such as about 30 nucleotides) .
  • the effector protein cleaves a labeled detector RNA to generate a signal (e.g., an increased signal or a decreased signal) thereby allowing for the qualitative and quantitative detection of the target RNA in the sample.
  • a signal e.g., an increased signal or a decreased signal
  • the specific detection and quantification of RNA in the sample allows for a multitude of applications including diagnostics.
  • the methods include contacting a sample with: i) an RNA guide (e.g., crRNA) and/or a nucleic acid encoding the RNA guide, wherein the RNA guide consists of a direct repeat sequence and a spacer sequence capable of hybridizing to the target RNA; (ii) an engineered Class 2 type VI Cas13 protein with enhanced collateral activity compared to wild-type Cas13, such as a subject engineered Type VI-E or VI-F CRISPR-Cas effector protein (Cas13e or Cas13f) and/or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNase activity and cleaves the labeled detector RNA; and b) measuring a detectable RNA guide (
  • the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
  • the labeled detector RNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair.
  • FRET fluorescence resonance energy transfer
  • an amount of detectable signal produced by the labeled detector RNA is decreased or increased.
  • the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.
  • a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
  • the labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof.
  • the methods include the multi-channel detection of multiple independent target RNAs in a sample (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more target RNAs) by using multiple engineered Cas13, such as the engineered Type VI-E and/or VI-F CRISPR-Cas (Cas13e and/or Cas13f) systems of the invention, each including a distinct orthologous effector protein and corresponding RNA guides, allowing for the differentiation of multiple target RNAs in the sample.
  • multiple engineered Cas13 such as the engineered Type VI-E and/or VI-F CRISPR-Cas (Cas13e and/or Cas13f) systems of the invention, each including a distinct orthologous effector protein and corresponding RNA guides, allowing for the differentiation of multiple target RNAs in the sample.
  • the methods include the multi-channel detection of multiple independent target RNAs in a sample, with the use of multiple instances of engineered Cas13, such as engineered Type VI-E and/or VI-F CRISPR-Cas systems of the invention, each containing an orthologous effector protein with differentiable collateral RNase substrates.
  • engineered Cas13 such as engineered Type VI-E and/or VI-F CRISPR-Cas systems of the invention.
  • In vitro proximity labeling techniques employ an affinity tag combined with, a reporter group, e.g., a photoactivatable group, to label polypeptides and RNAs in the vicinity of a protein or RNA of interest in vitro. After UV irradiation, the photoactivatable groups react with proteins and other molecules that are in close proximity to the tagged molecules, thereby labelling them. Labelled interacting molecules can subsequently be recovered and identified.
  • the CRISPR-associated proteins can for instance be used to target probes to selected RNA sequences.
  • the CRISPR systems e.g., CRISPR-associated proteins
  • CRISPR-associated proteins can be used to isolate and/or purify the RNA.
  • the CRISPR-associated proteins can be fused to an affinity tag that can be used to isolate and/or purify the RNA-CRISPR-associated protein complex. These applications are useful, e.g., for the analysis of gene expression profiles in cells.
  • the CRISPR-associated proteins can be used to target a specific noncoding RNA (ncRNA) thereby blocking its activity.
  • ncRNA noncoding RNA
  • the CRISPR-associated proteins can be used to specifically enrich a particular RNA (including but not limited to increasing stability, etc. ) , or alternatively, to specifically deplete a particular RNA (e.g., particular splice variants, isoforms, etc. ) .
  • the CRISPR systems described herein can be used for preparing next generation sequencing (NGS) libraries.
  • NGS next generation sequencing
  • the CRISPR systems can be used to disrupt the coding sequence of a target gene product, and the CRISPR-associated protein transfected clones can be screened simultaneously by next-generation sequencing (e.g., on the Ion Torrent PGM system) .
  • next-generation sequencing e.g., on the Ion Torrent PGM system
  • next-generation sequencing e.g., on the Ion Torrent PGM system
  • a detailed description regarding how to prepare NGS libraries can be found, e.g., in Bell et al., “A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing, ” BMC Genomics, 15.1 (2014) : 1002, which is incorporated herein by reference in its entirety.
  • Microorganisms e.g., E. coli, yeast, and microalgae
  • E. coli, yeast, and microalgae are widely used for synthetic biology.
  • the development of synthetic biology has a wide utility, including various clinical applications.
  • the programmable CRISPR systems can be used to split proteins of toxic domains for targeted cell death, e.g., using cancer-linked RNA as target transcript.
  • pathways involving protein-protein interactions can be influenced in synthetic biological systems with, e.g., fusion complexes with the appropriate effectors such as kinases or enzymes.
  • crRNAs that target phage sequences can be introduced into the microorganism.
  • the disclosure also provides methods of vaccinating a microorganism (e.g., a production strain) against phage infection.
  • the CRISPR systems provided herein can be used to engineer microorganisms, e.g., to improve yield or improve fermentation efficiency.
  • the CRISPR systems described herein can be used to engineer microorganisms, such as yeast, to generate biofuel or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars.
  • the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and/or to modify endogenous genes, which may interfere with the biofuel synthesis.
  • the CRISPR systems provided herein can be used to induce death or dormancy of a cell (e.g., a microorganism such as an engineered microorganism) .
  • a cell e.g., a microorganism such as an engineered microorganism
  • These methods can be used to induce dormancy or death of a multitude of cell types including prokaryotic and eukaryotic cells, including, but not limited to mammalian cells (e.g., cancer cells, or tissue culture cells) , protozoans, fungal cells, cells infected with a virus, cells infected with an intracellular bacteria, cells infected with an intracellular protozoan, cells infected with a prion, bacteria (e.g., pathogenic and non-pathogenic bacteria) , protozoans, and unicellular and multicellular parasites.
  • mammalian cells e.g., cancer cells, or tissue culture cells
  • protozoans e.g., fun
  • engineered microorganisms e.g., bacteria
  • the systems described herein can be used as “kill-switches” to regulate and/or prevent the propagation or dissemination of an engineered microorganism.
  • the systems described herein can also be used in applications where it is desirable to kill or control a specific microbial population (e.g., a bacterial population) .
  • the systems described herein may include an RNA guide (e.g., a crRNA) that targets a nucleic acid (e.g., an RNA) that is genus-, species-, or strain-specific, and can be delivered to the cell.
  • a RNA guide e.g., a crRNA
  • a nucleic acid e.g., an RNA
  • the methods comprise contacting the cell with a system described herein including a Type VI-E and/or VI-F CRISPR-Cas effector proteins or a nucleic acid encoding the effector protein, and a RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the spacer sequence is complementary to at least 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides) of a target nucleic acid (e.g., a genus-, strain-, or species-specific RNA guide) .
  • a target nucleic acid e.g., a genus-, strain-, or species-specific RNA guide
  • the cleavage of non-target RNA by the Type VI-E and/or VI-F CRISPR-Cas effector proteins may induce programmed cell death, cell toxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, a reduction of cell growth, or a reduction in cell proliferation.
  • the cleavage of non-target RNA by the Type VI-E and/or VI-F CRISPR-Cas effector proteins may be bacteriostatic or bactericidal.
  • the CRISPR systems described herein have a wide variety of utility in plants.
  • the CRISPR systems can be used to engineer transcriptome of plants (e.g., improving production, making products with desired post-translational modifications, or introducing genes for producing industrial products) .
  • the CRISPR systems can be used to introduce a desired trait to a plant (e.g., without heritable modifications to the genome) , or regulate expression of endogenous genes in plant cells or whole plants.
  • the CRISPR systems can be used to identify, edit, and/or silence genes encoding specific proteins, e.g., allergenic proteins (e.g., allergenic proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans) .
  • allergenic proteins e.g., allergenic proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans
  • a detailed description regarding how to identify, edit, and/or silence genes encoding proteins is described, e.g., in Nicolaou et al., “Molecular diagnosis of peanut and legume allergy, ” Curr. Opin. Allergy Clin. Immunol. 11 (3) : 222-8, 2011, and WO 2016205764 A1; both of which are incorporated herein by reference in the entirety.
  • pooled CRISPR screening is a powerful tool for identifying genes involved in biological mechanisms such as cell proliferation, drug resistance, and viral infection.
  • Cells are transduced in bulk with a library of guide RNA (gRNA) -encoding vectors described herein, and the distribution of gRNAs is measured before and after applying a selective challenge.
  • gRNA guide RNA
  • Pooled CRISPR screens work well for mechanisms that affect cell survival and proliferation, and they can be extended to measure the activity of individual genes (e.g., by using engineered reporter cell lines) .
  • Arrayed CRISPR screens in which only one gene is targeted at a time, make it possible to use RNA-seq as the readout.
  • the CRISPR systems as described herein can be used in single-cell CRISPR screens.
  • a detailed description regarding pooled CRISPR screenings can be found, e.g., in Datlinger et al., “Pooled CRISPR screening with single-cell transcriptome read-out, ” Nat. Methods. 14 (3) : 297-301, 2017, which is incorporated herein by reference in its entirety.
  • the CRISPR systems described herein can be used for in situ saturating mutagenesis.
  • a pooled guide RNA library can be used to perform in situ saturating mutagenesis for particular genes or regulatory elements.
  • Such methods can reveal critical minimal features and discrete vulnerabilities of these genes or regulatory elements (e.g., enhancers) . These methods are described, e.g., in Canver et al., “BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis, ” Nature 527 (7577) : 192-7, 2015, which is incorporated herein by reference in its entirety.
  • the CRISPR systems described herein can have various RNA-related applications, e.g., modulating gene expression, degrading a RNA molecule, inhibiting RNA expression, screening RNA or RNA products, determining functions of lincRNA or non-coding RNA, inducing cell dormancy, inducing cell cycle arrest, reducing cell growth and/or cell proliferation, inducing cell anergy, inducing cell apoptosis, inducing cell necrosis, inducing cell death, and/or inducing programmed cell death.
  • WO 2016/205764 A1 which is incorporated herein by reference in its entirety.
  • the methods described herein can be performed in vitro, in vivo, or ex vivo.
  • the CRISPR systems described herein can be administered to a subject having a disease or disorder to target and induce cell death in a cell in a diseased state (e.g., cancer cells or cells infected with an infectious agent) .
  • a diseased state e.g., cancer cells or cells infected with an infectious agent
  • the CRISPR systems described herein can be used to target and induce cell death in a cancer cell, wherein the cancer cell is from a subject having a Wilms′ tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leuk
  • the CRISPR systems described herein can be used to modulate gene expression.
  • the CRISPR systems can be used, together with suitable guide RNAs, to target gene expression, via control of RNA processing.
  • the control of RNA processing can include, e.g., RNA processing reactions such as RNA splicing (e.g., alternative splicing) , viral replication, and tRNA biosynthesis.
  • the RNA targeting proteins in combination with suitable guide RNAs can also be used to control RNA activation (RNAa) .
  • RNA activation is a small RNA-guided and Argonaute (Ago) -dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the transcriptional/epigenetic level.
  • RNAa leads to the promotion of gene expression, so control of gene expression may be achieved that way through disruption or reduction of RNAa.
  • the methods include the use of the RNA targeting CRISPR as substitutes for e.g., interfering ribonucleic acids (such as siRNAs, shRNAs, or dsRNAs) .
  • interfering ribonucleic acids such as siRNAs, shRNAs, or dsRNAs
  • the methods of modulating gene expression are described, e.g., in WO 2016205764, which is incorporated herein by reference in its entirety.
  • the target RNAs can include interfering RNAs, i.e., RNAs involved in the RNA interference pathway, such as small hairpin RNAs (shRNAs) , small interfering (siRNAs) , etc.
  • the target RNAs include, e.g., miRNAs or double stranded RNAs (dsRNA) .
  • RNA targeting protein and suitable guide RNAs are selectively expressed (for example spatially or temporally under the control of a regulated promoter, for example a tissue-or cell cycle-specific promoter and/or enhancer) , this can be used to protect the cells or systems (in vivo or in vitro) from RNA interference (RNAi) in those cells.
  • a regulated promoter for example a tissue-or cell cycle-specific promoter and/or enhancer
  • RNAi RNA interference
  • This may be useful in neighboring tissues or cells where RNAi is not required or for the purposes of comparison of the cells or tissues where the CRISPR-associated proteins and suitable crRNAs are and are not expressed (i.e., where the RNAi is not controlled and where it is, respectively) .
  • RNA targeting proteins can be used to control or bind to molecules comprising or consisting of RNAs, such as ribozymes, ribosomes, or riboswitches.
  • the guide RNAs can recruit the RNA targeting proteins to these molecules so that the RNA targeting proteins are able to bind to them.
  • Riboswitches are regulatory segments of messenger RNAs that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules.
  • a specific riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene.
  • the riboswitch activity can be controlled by the use of the RNA targeting proteins in combination with suitable guide RNAs to target the riboswitches. This may be achieved through cleavage of, or binding to, the riboswitch.
  • the CRISPR-associated proteins described herein can be fused to a base-editing domain, such as ADAR1, ADAR2, APOBEC, or activation-induced cytidine deaminase (AID) , and can be used to modify an RNA sequence (e.g., an mRNA) .
  • the CRISPR-associated protein includes one or more mutations (e.g., in a catalytic domain) , which renders the subject CRISPR-associated protein incapable of cleaving RNA (e.g., the dCas13 version of the engineered Class 2 type VI Cas13 protein described herein) .
  • RNA-binding fusion polypeptide comprising a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID) fused to an RNA-binding domain, such as MS2 (also known as MS2 coat protein) , Qbeta (also known as Qbeta coat protein) , or PP7 (also known as PP7 coat protein) .
  • MS2 also known as MS2 coat protein
  • Qbeta also known as Qbeta coat protein
  • PP7 also known as PP7 coat protein
  • MS2 (MS2 coat protein)
  • the RNA binding domain can bind to a specific sequence (e.g., an aptamer sequence) or secondary structure motifs on a crRNA of the system described herein (e.g., when the crRNA is in an effector-crRNA complex) , thereby recruiting the RNA binding fusion polypeptide (which has a base-editing domain) to the effector complex.
  • a specific sequence e.g., an aptamer sequence
  • secondary structure motifs on a crRNA of the system described herein (e.g., when the crRNA is in an effector-crRNA complex) , thereby recruiting the RNA binding fusion polypeptide (which has a base-editing domain) to the effector complex.
  • the CRISPR system includes a CRISPR associated protein, a crRNA having an aptamer sequence (e.g., an MS2 binding loop, a QBeta binding loop, or a PP7 binding loop) , and a RNA-binding fusion polypeptide having a base-editing domain fused to an RNA-binding domain that specifically binds to the aptamer sequence.
  • the CRISPR-associated protein forms a complex with the crRNA having the aptamer sequence.
  • the RNA-binding fusion polypeptide binds to the crRNA (via the aptamer sequence) thereby forming a tripartite complex that can modify a target RNA.
  • an inactivated or dCas13 version of the engineered Class 2 type VI Cas13 protein substantially lacking collateral activity described herein can be used to target and bind to specific splicing sites on RNA transcripts. Binding of the inactivated CRISPR-associated protein to the RNA may sterically inhibit interaction of the spliceosome with the transcript, enabling alteration in the frequency of generation of specific transcript isoforms. Such method can be used to treat disease through exon skipping such that an exon having a mutation may be skipped in a mature protein.
  • the CRISPR systems described herein can have various therapeutic applications. Such applications may be based on one or more of the abilities below, both in vitro and in vivo, of the subject engineered Cas13, e.g., engineered CRISPR/Cas13e or Cas13f systems: induce cellular senescence, induce cell cycle arrest, inhibit cell growth and/or proliferation, induce apoptosis, induce necrosis, etc.
  • engineered Cas13 e.g., engineered CRISPR/Cas13e or Cas13f systems: induce cellular senescence, induce cell cycle arrest, inhibit cell growth and/or proliferation, induce apoptosis, induce necrosis, etc.
  • the new engineered CRISPR systems can be used to treat various diseases and disorders, e.g., genetic disorders (e.g., monogenetic diseases) , diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne Muscular Dystrophy (DMD) , BCL11a targeting) , and various cancers, etc.
  • diseases and disorders e.g., genetic disorders (e.g., monogenetic diseases) , diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne Muscular Dystrophy (DMD) , BCL11a targeting) , and various cancers, etc.
  • the CRISPR systems described herein can be used to edit a target nucleic acid to modify the target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleic acid residues) .
  • the CRISPR systems described herein can be used for treating a disease caused by overexpression of RNAs, toxic RNAs, and/or mutated RNAs (e.g., splicing defects or truncations) .
  • expression of toxic RNAs may be associated with the formation of nuclear inclusions and late-onset degenerative changes in brain, heart, or skeletal muscle.
  • the disorder is myotonic dystrophy. In myotonic dystrophy, the main pathogenic effect of the toxic RNAs is to sequester binding proteins and compromise the regulation of alternative splicing (see, e.g., Osborne et al., “RNA-dominant diseases, ” Hum. Mol. Genet., 2009 Apr.
  • DM dystrophia myotonica
  • UTR 3’-untranslated region
  • DMPK a gene encoding a cytosolic protein kinase.
  • the CRISPR systems as described herein can target overexpressed RNA or toxic RNA, e.g., the DMPK gene or any of the mis-regulated alternative splicing in DM1 skeletal muscle, heart, or brain.
  • the CRISPR systems described herein can also target trans-acting mutations affecting RNA-dependent functions that cause various diseases such as, e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA) , and Dyskeratosis congenita.
  • diseases e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA) , and Dyskeratosis congenita.
  • SMA Spinal muscular atrophy
  • Dyskeratosis congenita e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA) , and Dyskeratosis congenita.
  • SMA Spinal muscular atrophy
  • Dyskeratosis congenita Dyskeratosis congenita.
  • a list of diseases that can be treated using the CRISPR systems described herein is summarized in Cooper et al., “RNA and disease, ” Cell, 136.4 (2009) : 777-793, and WO 2016/2057
  • the CRISPR systems described herein can also be used in the treatment of various tauopathies, including, e.g., primary and secondary tauopathies, such as primary age-related tauopathy (PART) /Neurofibrillary tangle (NFT) -predominant senile dementia (with NFTs similar to those seen in Alzheimer Disease (AD) , but without plaques) , dementia pugilistica (chronic traumatic encephalopathy) , and progressive supranuclear palsy.
  • PART primary age-related tauopathy
  • NFT Neurofibrillary tangle
  • a useful list of tauopathies and methods of treating these diseases are described, e.g., in WO 2016205764, which is incorporated herein by reference in its entirety.
  • the CRISPR systems described herein can also be used to target mutations disrupting the cis-acting splicing codes that can cause splicing defects and diseases.
  • diseases include, e.g., motor neuron degenerative disease that results from deletion of the SMN1 gene (e.g., spinal muscular atrophy) , Duchenne Muscular Dystrophy (DMD) , frontotemporal dementia, and Parkinsonism linked to chromosome 17 (FTDP-17) , and cystic fibrosis.
  • the CRISPR systems described herein can further be used for antiviral activity, in particular against RNA viruses.
  • the CRISPR-associated proteins can target the viral RNAs using suitable guide RNAs selected to target viral RNA sequences.
  • the CRISPR systems described herein can also be used to treat a cancer in a subject (e.g., a human subject) .
  • the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule that is aberrant (e.g., comprises a point mutation or are alternatively-spliced) and found in cancer cells to induce cell death in the cancer cells (e.g., via apoptosis) .
  • the CRISPR systems described herein can also be used to treat an autoimmune disease or disorder in a subject (e.g., a human subject) .
  • a subject e.g., a human subject
  • the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule that is aberrant (e.g., comprises a point mutation or are alternatively-spliced) and found in cells responsible for causing the autoimmune disease or disorder.
  • the CRISPR systems described herein can also be used to treat an infectious disease in a subject.
  • the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule expressed by an infectious agent (e.g., a bacteria, a virus, a parasite or a protozoan) in order to target and induce cell death in the infectious agent cell.
  • an infectious agent e.g., a bacteria, a virus, a parasite or a protozoan
  • the CRISPR systems may also be used to treat diseases where an intracellular infectious agent infects the cells of a host subject. By programming the CRISPR-associated protein to target a RNA molecule encoded by an infectious agent gene, cells infected with the infectious agent can be targeted and cell death induced.
  • RNA sensing assays can be used to detect specific RNA substrates.
  • the CRISPR-associated proteins can be used for RNA-based sensing in living cells. Examples of applications are diagnostics by sensing of, for examples, disease-specific RNAs.
  • the methods of the invention can be used to introduce the CRISPR systems described herein into a cell, and cause the cell and/or its progeny to alter the production of one or more cellular produces, such as antibody, starch, ethanol, or any other desired products.
  • Such cells and progenies thereof are within the scope of the invention.
  • the methods and/or the CRISPR systems described herein lead to modification of the translation and/or transcription of one or more RNA products of the cells.
  • the modification may lead to increased transcription/translation/expression of the RNA product.
  • the modification may lead to decreased transcription/translation/expression of the RNA product.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (aprimary human cell or an established human cell line) .
  • the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey) , a cow/bull/cattle, sheep, goat, pig, horse, dog, cat, rodent (such as rabbit, mouse, rat, hamster, etc) .
  • the cell is from fish (such as salmon) , bird (such as poultry bird, including chick, duck, goose) , reptile, shellfish (e.g., oyster, claim, lobster, shrimp) , insect, worm, yeast, etc.
  • the cell is from a plant, such as monocot or dicot.
  • the plant is a food crop such as barley, cassava, cotton, groundnuts or peanuts, maize, millet, oil palm fruit, potatoes, pulses, rapeseed or canola, rice, rye, sorghum, soybeans, sugar cane, sugar beets, sunflower, and wheat.
  • the plant is a cereal (barley, maize, millet, rice, rye, sorghum, and wheat) .
  • the plant is a tuber (cassava and potatoes) .
  • the plant is a sugar crop (sugar beets and sugar cane) .
  • the plant is an oil-bearing crop (soybeans, groundnuts or peanuts, rapeseed or canola, sunflower, and oil palm fruit) .
  • the plant is a fiber crop (cotton) .
  • the plant is a tree (such as a peach or a nectarine tree, an apple or pear tree, a nut tree such as almond or walnut or pistachio tree, or a citrus tree, e.g., orange, grapefruit or lemon tree) , a grass, a vegetable, a fruit, or an algae.
  • a tree such as a peach or a nectarine tree, an apple or pear tree, a nut tree such as almond or walnut or pistachio tree, or a citrus tree, e.g., orange, grapefruit or lemon tree
  • the plant is a nightshade plant; a plant of the genus Brassica; a plant of the genus Lactuca; a plant of the genus Spinacia; a plant of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.
  • a related aspect provides cells or progenies thereof modified by the methods of the invention using the CRISPR systems described herein.
  • the cell is modified in vitro, in vivo, or ex vivo.
  • the cell is a stem cell.
  • the CRISPR systems described herein comprising an engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity (such as Cas13e or Cas13f) , or any of the components thereof described herein (Cas13 proteins, derivatives, functional fragments or the various fusions or adducts thereof, and guide RNA/crRNA) , nucleic acid molecules thereof, and/or nucleic acid molecules encoding or providing components thereof, can be delivered by various delivery systems such as vectors, e.g., plasmids and viral delivery vectors, using any suitable means in the art. Such methods include (and are not limited to) electroporation, lipofection, microinjection, transfection, sonication, gene gun, etc.
  • the CRISPR-associated proteins and/or any of the RNAs (e.g., guide RNAs or crRNAs) and/or accessory proteins can be delivered using suitable vectors, e.g., plasmids or viral vectors, such as adeno-associated viruses (AAV) , lentiviruses, adenoviruses, retroviral vectors, and other viral vectors, or combinations thereof.
  • suitable vectors e.g., plasmids or viral vectors, such as adeno-associated viruses (AAV) , lentiviruses, adenoviruses, retroviral vectors, and other viral vectors, or combinations thereof.
  • the proteins and one or more crRNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors.
  • the nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to the bacteria using a phage.
  • Exemplary phages include, but are not limited to, T4 phage, Mu, ⁇ phage, T5 phage, T7 phage, T3 phage, ⁇ 29, M13, MS2, Q ⁇ , and ⁇ X174.
  • the vectors e.g., plasmids or viral vectors
  • the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration.
  • Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
  • the delivery is via adenoviruses, which can be at a single dose containing at least 1 ⁇ 10 5 particles (also referred to as particle units, pu) of adenoviruses.
  • the dose preferably is at least about 1 ⁇ 10 6 particles, at least about 1 ⁇ 10 7 particles, at least about 1 ⁇ 10 8 particles, and at least about 1 ⁇ 10 9 particles of the adenoviruses.
  • the delivery methods and the doses are described, e.g., in WO 2016205764 A1 and U.S. Pat. No. 8,454,972 B2, both of which are incorporated herein by reference in the entirety.
  • the delivery is via plasmids.
  • the dosage can be a sufficient number of plasmids to elicit a response.
  • suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg.
  • Plasmids will generally include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR-associated proteins and/or an accessory protein, each operably linked to a promoter (e.g., the same promoter or a different promoter) ; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii) .
  • the plasmids can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on different vectors.
  • the frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian) , or a person skilled in the art.
  • the delivery is via liposomes or lipofection formulations and the like, and can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Pat. Nos. 5,593,972; 5,589,466; and 5,580,859; each of which is incorporated herein by reference in its entirety.
  • the delivery is via nanoparticles or exosomes.
  • exosomes have been shown to be particularly useful in delivery RNA.
  • CRISPR-associated proteins are linked to the CRISPR-associated proteins.
  • the CRISPR-associated proteins and/or guide RNAs are coupled to one or more CPPs to effectively transport them inside cells (e.g., plant protoplasts) .
  • the CRISPR-associated proteins and/or guide RNA (s) are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cell delivery.
  • CPPs are short peptides of fewer than 35 amino acids derived either from proteins or from chimeric sequences capable of transporting biomolecules across cell membrane in a receptor independent manner.
  • CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having proline-rich and anti-microbial sequences, and chimeric or bipartite peptides.
  • CPPs include, e.g., Tat (which is a nuclear transcriptional activator protein required for viral replication by HIV type 1) , penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin ⁇ 3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide.
  • Tat which is a nuclear transcriptional activator protein required for viral replication by HIV type 1
  • FGF Kaposi fibroblast growth factor
  • FGF Kaposi fibroblast growth factor
  • integrin ⁇ 3 signal peptide sequence integrin ⁇ 3 signal peptide sequence
  • polyarginine peptide Args sequence e.g., in et al., “Prediction of cell-penetrating peptides, ” Methods Mol.
  • kits comprising any two or more components of the subject CRISPR/Cas system described herein comprising an engineered Class 2 type VI Cas13 protein, such as those either substantially lacking or having enhanced collateral activity, such as the Cas13e and Cas13f proteins, derivatives, functional fragments or the various fusions or adducts thereof, guide RNA/crRNA, complexes thereof, vectors encompassing the same, or host encompassing the same.
  • an engineered Class 2 type VI Cas13 protein such as those either substantially lacking or having enhanced collateral activity, such as the Cas13e and Cas13f proteins, derivatives, functional fragments or the various fusions or adducts thereof, guide RNA/crRNA, complexes thereof, vectors encompassing the same, or host encompassing the same.
  • the kit further comprises an instruction to use the components encompassed therein, and/or instructions for combining with additional components that may be available elsewhere.
  • the kit further comprises one or more nucleotides, such as nucleotide (s) corresponding to those useful to insert the guide RNA coding sequence into a vector and operably linking the coding sequence to one or more control elements of the vector.
  • nucleotides such as nucleotide (s) corresponding to those useful to insert the guide RNA coding sequence into a vector and operably linking the coding sequence to one or more control elements of the vector.
  • the kit further comprises one or more buffers that may be used to dissolve any of the components, and/or to provide suitable reaction conditions for one or more of the components.
  • buffers may include one or more of PBS, HEPES, Tris, MOPS, Na 2 CO 3 , NaHCO 3 , NaB, or combinations thereof.
  • the reaction condition includes a proper pH, such as a basic pH. In certain embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container.
  • This example demonstrates that collateral effect or non-sequence-specific endonuclease activity of the Cas13 enzymes (e.g., Cas13e) can be largely reduced by introducing mutations that reduce the affinity between Cas13e and potential RNA targets (sequence specific or non-sequence specific targets) , thus disproportionally reducing collateral non-sequence-specific endonuclease activity, while substantially maintaining sequence-specific endonuclease activity against the target RNA, partly due to the binding between the guide sequence and the target RNA. See FIG. 1.
  • sequences that are spatially close to the two HEPN domains in Cas13e were systematically mutated (see FIG. 3) over the entire regions of interest.
  • mutations were focused on those residues that likely participate in RNA binding (or RNA binding hotspots) , namely those with nitrogen-containing and/or positively charged side chain groups such as R, K, H, N, or Q residues.
  • RNA binding or RNA binding hotspots
  • nitrogen-containing and/or positively charged side chain groups such as R, K, H, N, or Q residues.
  • a BpiI recognition sequence was introduced, i.e., GTCTTC on one end (corresponding to the di-peptide sequence of ValPhe or VF) , and GAAGAC on the other end (corresponding to the di-peptide sequence of GluAsp or ED) .
  • GTCTTC corresponding to the di-peptide sequence of ValPhe or VF
  • GAAGAC corresponding to the di-peptide sequence of GluAsp or ED
  • 5-8 mutations were introduced between each pair of BpiI recognition sequences.
  • Y/S/T>A style mutants were introduced.
  • an EGFP-mCherry double fluorescent reporting system was constructed (see FIG. 4) .
  • expression of EGFP and mCherry were under the separate but identical control of their respective SV40 promoters, in order to ensure that their mRNA ratio was relatively stably maintained in transfected cells.
  • the gRNA of this system specifically targeted EGFP coding sequence (mRNA) .
  • each tested engineered Cas13e has a NLS (nuclear localization sequence) at the N-terminus, as well as the C-terminus.
  • the CMV promoter was used to drive the expression of the engineered Cas13e.
  • the sequences of the EGFP and mCherry reporters are in SEQ ID NOs: 1 and 2.
  • the gRNA is SEQ ID NO: 3.
  • Wild type Cas13e protein is SEQ ID NO: 4, and its codon-optimized polynucleotide coding sequence is SEQ ID NO: 5.
  • Human HEK293T cells were cultured in 24-well tissue culture plates according to standard methods, before the double-fluorescent reporting system plasmid was transfected into the cells using standard polyethylenimine (PEI) transfection. Transfected cells were then cultured at 37°C under CO 2 for 48 hrs. EGFP and mCherry signals were detected using FACS.
  • PEI polyethylenimine
  • mutant/engineered Cas13e has similar/equivalent EGFP signal compared to the wild-type Cas13e, indicating that the guide-sequence-specific cleavage of the target RNA (EGFP) was not/little affected by the mutations in the engineered Cas13e;
  • mutant/engineered Cas13e has similar/equivalent mCherry signal compared to the nuclease dead dCas13e, indicating that the non-sequence-specific cleavage of the non-target RNA (mCherry) was non-existing in the engineered Cas13e, just like dCas13e that is unable to cleave mCherry mRNA.
  • Mut-17 and Mut-19 essentially eliminated collateral effect of wild-type Cas13c, while maintained relatively high guide-sequence specific endonuclease activity.
  • the method described herein has been shown to be able to identify residues for engineering even though these residues are far away from the HEPN domains in primary sequence, but can be shown to be spatially close to the HEPN domains based on predicted 3D structure (using commonly available tools such as PyMOL or I-TASSER) . See FIG. 8.
  • M17.0-6 is the same as Mut-17.
  • point mutations M17.6, M17.8 and M17.9 SEQ ID NOs: 37-39 essentially eliminated collateral effect of wild-type Cas13e to dCas13e. 1 level, while the other point mutations retained different degrees of collateral effect compared to wild-type Cas13e. 1, including in some cases enhanced collateral effect (see FIG. 10) . Therefore, residues Y672 and Y676 in the Mut-17 region of wtCas13e. 1 appear to be two key residues that affect the collateral circumcision effect of wild-type Cas13e. 1.
  • point mutations M19.2 and M19.5 (SEQ ID NOs: 45 and 48) essentially eliminated collateral effect of wild-type Cas13e to dCas13e. 1 level, while the other point mutations retained different degrees of collateral effect compared to wild-type Cas13e. 1 (see FIG. 13) . Therefore, residues Y715 in the Mut-19 region of wtCas13e. 1 appear to be a key residue that affects the collateral circumcision effect of wild-type Cas13e. 1.
  • RNA degradation by the Cas13 family of effector enzymes has previously been found in glioma cells and flies, but its presence in mammalian cells has not been definitively demonstrated.
  • this example demonstrates that Cas13 could indeed induce substantial collateral effects in HEK293T cells when targeting either exogenous and endogenous genes.
  • Cas13d was shown to mediate transcriptome-wide RNA off-target editing, causing cell growth arrest and reducing cell viability.
  • Cas13 (Cas13a or Cas13d) were co-transfected with EGFP and mCherry coding sequences, together with targeted (against mCherry) or non-targeted (NT, control) guide RNA (gRNA) into HEK293T cells.
  • targeted mCherry or non-targeted guide RNA (gRNA)
  • gRNA guide RNA
  • collateral effects are not limited to transiently overexpressed exogenous genes.
  • the data presented herein also demonstrates that Cas13d could induce collateral effects when targeting endogenous genes in HEK293T.
  • an unbiased screening system was designed based on the dual-fluorescence approach described above, in which coding sequences for EGFP, mCherry, EGFP-targeting gRNA, together with each Cas13 variants, were inserted into one plasmid for expression in 293T cells.
  • expression of EGFP and expression of mCherry were driven by the same SV40 promoter, in order to ensure roughly equally stable expression of the reporter genes in the transfected host cell.
  • the gRNA was chosen to be specific for EGFP mRNA.
  • Each coding sequence for Cad13d and variants has an N-terminal and a C-terminal nuclear localization signal (NLS) , and expression of Cas13d and variants /mutants was driven by the strong CAG promoter.
  • the EGFP coding sequence is:
  • the mCherry coding sequence is:
  • the corresponding DNA sequence of the gRNA is:
  • the wild-type Cas13d protein sequence is SEQ ID NO: Cas13d:
  • the coding sequence for the wild-type Cas13d is:
  • the CAG promoter sequence is:
  • the SV40 promoter sequence is:
  • the HEPN1-I, HEPN1-II, and HEPN2 domains of Cas13d were chosen for generating a Cas13d mutagenesis library.
  • these regions were divided into 21 small segments (N1-N21) , each with about 36 residues. More specifically, these 21 mutated regions cover HEPN1-I (N1-N6) , HEPN1-II (N8-N10) , HEPN2 (N14-N21) , Helical-1 (N7) and Helical-2 (N10-N14) domains (FIG. 17C) .
  • a BpiI restriction enzyme recognition site (GTCTTC, corresponding to encoded residues VF; reverse complement GAAGAC, corresponding to encoded residues ED) was introduced at each end of the segments.
  • GTCTTC corresponding to encoded residues VF
  • GAAGAC reverse complement GAAGAC
  • ED encoded residues
  • these Cas13d mutants were functionally screened to assess their collateral vs. gRNA-guided cleavage activities.
  • human HEK293 cells were grown in 24-well tissue culture plates to a suitable density before the cells were transfected with PEI reagents and plasmids that express each mutant Cas13d and the reporter system fluorescent proteins.
  • Transfected cells were cultured at 37°C in incubator under 5%CO 2 for about 48 hours, before measuring EGFP and mCherry signals in the cells with FACS.
  • Mutants leading to low percentage of the gRNA-targeted EGFP signal (lower percentage of EGFP + cells, as a readout for preserved gRNA-guided cleavage) and high percentage of non-targeted mCherry signal (higher percentage of mCherry + cells, as a readout for lacking collateral effect) were selected.
  • dCas13d with no gRNA-guided cleavage was used as a negative control, and the results (mean ⁇ s.e.m. ) were normalized against that of dCas13d and listed below.
  • Cas13d mutants located at the upper left area of FIG. 17D had low collateral effect (high mCherry signal) and high gRNA-guided cleavage activity (low EGFP signal) , and were selected as the desired low /no collateral effect mutants.
  • these mutants exhibited less than 27.5%collateral effect (e.g., ⁇ 72.5%mCherry + cells) , and ⁇ 75%gRNA-guided cleavage ( ⁇ 25%EGFP + cells) . They include: N1V7, N2V7, N2V8, N3V7, and N15V4, etc. (see above table and FIG. 17D) . Based on FACS data (not shown) , these mutants have significantly reduced collateral effect compared to wild-type.
  • some of the Cas13d mutants exhibited low collateral effect (e.g., ⁇ 27.5%collateral effect, or ⁇ 72.5%mCherry + cells) , and intermediate gRNA-guided cleavage (e.g., 25% ⁇ EGFP + cells ⁇ 75%) , including: N2V4, N2V5, N4V3, N6V3, N10V6, N15V2, N20V6, and N20-Y910A, etc. (see above table and FIG. 17D) .
  • the gRNA-guided cleavage efficiency for these mutants can be enhanced further by, for example, using multiple gRNA targeting different sites of the target sequence, and the collateral effect would remain low.
  • mutants having substantially retained (e.g., retaining at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) wild-type level gRNA-guided cleavage, while substantially reducing /eliminating (at least about 72.5%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) Cas13d collateral effect.
  • N2V7 and N2V8 retained relatively high guide RNA-specific cleavage, with essentially eliminated Cas13d collateral effect, and the residues affected by these mutants are very close together, further mutagenesis study in the two regions of these mutants was conducted, by generating a number of additional mutants with single, double, triple, or quadruple combination mutations.
  • the sequences of these mutants and the corresponding wild-type sequences (N2C) are listed below:
  • mutants occupying the upper left corner of FIG. 17E were selected.
  • N2V8 (carrying A134V, A140V, A141V, A143V) was believed to has superior characteristics, in that it retained relatively high guide RNA-specific cleavage, while essentially eliminated Cas13d collateral effect. See data above and FIGs. 17D and 17E.
  • This mutant is sometimes referred to as cfCas13d (collateral free Cas13d) for further functional characterization.
  • mutations are mainly located within the HEPN1-1 domain (e.g., residues 90-292) , Helical2 domain (e.g., residues 536-690) , and the HEPN2 domain (e.g., residues 690-967 in Cas13d) .
  • substitutions by residues other than Ala are similarly effective to reduce /eliminate collateral effect.
  • mutants with significantly enhanced collateral effect, based on ⁇ 87.5%collateral cleavage efficiency (e.g., ⁇ 12.5% mCherry + cells) and better gRNA-guided cleavage compared to wild-type (e.g., ⁇ 4%EGFP + cells) .
  • mutants include: N2-Y142A, N4-Y193A, N12-Y604A, N21V7, etc.
  • N2-Y142A is located in the Helical2 domain, extending towards the two HEPN domains in the 3D structure.
  • N4-Y193A and N21V7 are within the HEPN1 and HEPN2 domains, respectively, and are relatively far away from the catalytic active site.
  • the residues involved in these mutants are listed below.
  • This example provides additional Cas13e mutants with reduced /eliminated collateral effect, based on knowledge of Cas13d mutants screening and simulated structural analysis of Cas13e (see FIG. 19A) .
  • a mutagenesis library was developed for Cas13e, covering HEPN1 and HEPN2 domains (FIG. 19B) . At least 90 different mutants were constructed, each comprising 1- 5 amino acid residue changes compared to the wild-type sequence.
  • the various Cas13e mutants and the corresponding wild-type sequences (M1-M21) are listed below.
  • these Cas13e mutants were functionally screened to assess their collateral vs. gRNA-guided cleavage activities.
  • human HEK293 cells were grown in 24-well tissue culture plates to a suitable density before the cells were transfccted with PEI reagents and plasmids that express each mutant Cas13e and the reporter system fluorescent proteins.
  • Transfected cells were cultured at 37°C in incubator under 5%CO2 for about 48 hours, before measuring EGFP and mCherry signals in the cells with FACS.
  • Mutants leading to low percentage of the gRNA-targeted EGFP signal (lower percentage of EGFP + cells, as a readout for preserved gRNA-guided cleavage) and high percentage of non-targeted mCherry signal (higher percentage of mCherry + cells, as a readout for lacking collateral effect) were selected.
  • dCas13e with no gRNA-guided cleavage was used as a negative control, and the results (mean ⁇ s.e.m. ) were normalized against that of dCas13e and listed below.
  • Cas13e mutants located at the upper left area of FIG. 19C had low collateral effect (high mCherry signal) and high gRNA-guided cleavage activity (low EGFP signal) , and were selected as the desired low /no collateral effect mutants.
  • Cas13e-M17YY (carrying Y672A, Y676A) exhibited similarly high level of EGFP knockdown and lower mCherry knockdown, compared with wild-type Cas13e (FIGs. 19C and 19D) .
  • these mutants exhibited less than 25%collateral effect (e.g., ⁇ 75%mCherry + cells) , and ⁇ 75%gRNA-guided cleavage ( ⁇ 25%EGFP + cells) .
  • They include: M1V4, M2V2, M2V3, M2V4, M5V1, M6V2, M6V3, M6V4, M7V1, M7V2, M7V3, M7-Y55A, M7-Y61A, M11V1, M12V3, M15V1, M15V2, M15-Y643A, M15-Y647A, M16V1, M16V2, M17V2, M18V2, M18V3, M19V2, M19V3, M19-IA, etc. (see above table and FIG. 19C) .
  • some of the Cas13e mutants exhibited low collateral effect (e.g., ⁇ 25%collateral effect, or ⁇ 75%mCherry + cells) , and intermediate gRNA-guided cleavage (e.g., 25% ⁇ EGFP + cells ⁇ 75%) , including: M17YY, M8V4, M9V1, M11V2, M11V3, M13V1, M13V2, M13V3, M15V3, M20V2, etc. (see above table and FIG. 19C) .
  • the gRNA-guided cleavage efficiency for these mutants can be enhanced further by, for example, using multiple gRNA targeting different sites of the target sequence, and the collateral effect would remain low.
  • mutants having substantially retained (e.g., retaining at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) wild-type level gRNA-guided cleavage, while substantially reducing /eliminating (at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) collateral effect.
  • residues in these regions may have participated in binding between Cas13e to the target RNA and/or the non-specific RNA, and mutations in these residues had different /differential effects on Cas13e affinity towards different RNA targets, hence the cleavage efficiency towards these RNA targets.
  • mutations are located within the HEPN1 domain and the inter-domain linker (IDL) region (e.g., residues 1-194 in Cas13e) , and the HEPN2 domain (e.g., residues 620-775 in Cas13e) .
  • IDL inter-domain linker
  • substitutions by residues other than Ala are similarly effective to reduce /eliminate collateral effect.
  • M17YY is sometimes referred to as cfCas13e (collateral free Cas13e) herein for further functional characterization.
  • mutants with significantly enhanced collateral effect, based on ⁇ 60%collateral cleavage efficiency (e.g., ⁇ 40%mCherry + cells) and better gRNA-guided cleavage compared to wild-type (e.g., ⁇ 5.5%EGFP + cells) .
  • mutants include: M14V2, M16V3, M18V1, M19-G712A, M19-T725A, M19-C727A, etc.
  • These mutants are mainly located between the two catalytic active sites formed by the RXXXXH motifs.
  • M14V2 is located in the Helical1-1 domain, around the beta-turn towards the two HEPN domains in the 3D structure.
  • M16V3, M18V1, M19-G712A, M19-T725A, and M19-C727A have mutations in the HEPN2 domain, around /near the alpha-helic and the its flanking unstructured regions, all close to the catalytic active site.
  • the residues involved in these mutants are listed below.
  • gRNA-1, g1 GTCCTCCTTGAAGTCGATGCCCTTCAGCTC
  • gRNA-2, g2 AGCACTGCACGCCGTAGGTCAGGGTGGTCA
  • gRNA-3, g3 GCAGGACCATGTGATCGCGCTTCTCGTTGG
  • gRNA-4, g4 GAACTTCAGGGTCAGCTTGCCGTAGGTGGC
  • the ssRNA target sequence and crRNA for determining gRNA-directed cleavage are:
  • ssRNA-cy5-Labled 5’-CY5-GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGU-BHQ2-3’
  • Cas13d-crRNA GGGCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU
  • the ssRNA target sequence and crRNA for determining collateral cleavage are:
  • ssRNA GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGU
  • Cas13d-crRNA GGGCAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU
  • RNA-FMA-Labeled FAM-AAAGAUACGAGGGUGCUAUGUUUCCACGCUCC-BHQ1
  • gRNA-1, g1 AGCACTGCACGCCGTAGGTCAGGGTGGTCA
  • gRNA-2, g2 GTCCTCCTTGAAGTCGATGCCCTTCAGCTC
  • gRNA-3, g3 TCGCCGTCCAGCTCGACCAGGATGGGCACC
  • gRNA-4, g4 TTCGGGCATGGCGGACTTGAAGAAGTCGTG
  • the ssRNA target sequence and crRNA for determining gRNA-directed cleavage are:
  • ssRNA-cy5-Labled 5’-CY5-GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGU-BHQ2-3’
  • Cas13e-crRNA AGUAGAUUGCUGUUCUACCAAGUAAUCCAUGCUGGAGCAGCCCCCGAUUUGUGGGGUGAUUACAGC
  • the ssRNA target sequence and crRNA for determining collateral cleavage are:
  • ssRNA GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGU
  • Cas13e-crRNA AGUAGAUUGCUGUUCUACCAAGUAAUCCAUGCUGGAGCAGCCCCCGAUUUGUGGGGUGAUUACAGC
  • RNA-FMA-Labeled FAM-AAAGAUACGAGGGUGCUAUGUUUCCACGCUCC-BHQ1
  • HEK293 cells were transfected with an all-in-one construct containing Cas13d, EGFP, mCherry, non-target (NT) gRNA, or a gRNA targeting each endogenous gene, and another construct containing BFP driven by CAG promoter. BFP was used here for normalizing transfection efficiency. About 48 hours post-transfection, the EGFP and mCherry fluorescence intensity was examined for the collateral effects and target transcript level for RNA knockdown activity (FIG. 20B) .
  • RNA interference activity by cfCas13d is still broadly applicable, cfCas13d and Cas13d were tested on randomly selected 14 endogenous transcripts in HEK293 cells. It was found that cfCas13d and Cas13d exhibited comparable efficient RNA knockdown activity (82 ⁇ 2%and 93 ⁇ 1%, respectively) , indicating that cfCas13d retained high-level activity of RNA interference on most endogenous genes (FIGs. 20H and 20I) .
  • RNA-seq transcriptome-wide RNA sequencing
  • cfCas13d Compared with Cas13d, cfCas13d remarkably reduced off-target changes when targeting RPL4 (down-regulated genes, 6750 vs. 39) , PPIA (9289 vs. 8) , CA2 (3519 vs. 18) , and PPARG (1601 vs. 52) .
  • cfCas13d could also target predicted gRNA-dependent off-target sites as Cas13d, indicating mutations in cfCas13d decrease collateral off-target cleavage but not gRNA-dependent off-target cleavage (FIGs.
  • Age-related macular degeneration (AMD) , a progressive condition that is untreatable in up to 90%of patients, is a leading cause of blindness in the elderly worldwide.
  • AMD Age-related macular degeneration
  • wet AMD affects only 10-15%of AMD patients, it emerges abruptly, and rapidly progresses to blindness if left untreated.
  • FDA-approved therapies A detailed understanding of the molecular mechanisms underlying wet AMD has led to several robust FDA-approved therapies.
  • CNV choroidal neovascularization
  • Aflibercept is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgG1 immunoglobulin.
  • VEGF vascular endothelial growth factor
  • Conbercept is a recombinant fusion protein composed of the second Ig domain of VEGFR1 and the third and fourth Ig domains of VEGFR2 to the constant region (Fc) of human IgG1.
  • This example utilizes a mouse model of wet AMD to show that cfCas13e, just like wild-type Cas13e, can efficiently knock down VEGFA to reduce CNV.
  • gRNA-1 g1
  • gRNA-2 g2
  • the corresponding DNA sequences of the gRNA are:
  • gRNA-1 (g1) : GTGCTGTAGGAAGCTCATCTCTCCTATGTG
  • gRNA-2 (g2) : GGTACTCCTGGAAGATGTCCACCAGGGTCT
  • coding sequence for cfCas13e (including two NLS sequences at the N-and C-terminus, under the EFS promoter) and the two gRNA's (g1+g2, under the control of the U6 promoter) were incorporated between the two ITR sequences of an AAV9 viral vector (with AAV9 serotype) .
  • Viral particles were injected directly into mouse subretinal space. After 21 days, laser light was used on the eyes of the experimental mouse to imitate UV-induced AMD. Seven days later, the extent of CNV in the experimental animals were determined (see FIGs. 19H and 19I) .
  • FIG. 19H expression of VEGFA target mRNA was normalized against untreated control animals. It is apparent that, when only a non-targeting (NT) guide RNA was provided, cfCase13e did not affect VEGFA expression. In contrast, when both g1 and g2 guide RNA's were provided, cfCas13e efficiently knocked down VEGFA expression to the same extent as the wild-type Cas13e, and to nearly undetectable level (FIG. 19H) .
  • NT non-targeting
  • the ITR sequence for the AAV9 viral vector is:
  • the nucleotide sequence of the EFS promoter used to drive cfCas13e expression is:
  • Applicant has designed, constructed, and obtained by screening numerous mutant Cas13 variants with reduced or eliminated collateral effect (as well as variants with enhanced collateral effects) .
  • the guide RNA-mediated functions of these Cas13e and Cas13d mutants /variants have been verified by in vitro biochemical reactions, endogenous gene expression knock down in mammalian cells, as well as gene therapy in an in vivo mouse model of AMD.
  • the Cas13d (CasRx) gene and gRNA backbone sequences were synthesized by a commercial source.
  • Vectors CAG-Cas13d-p2A-GFP and U6-DR-BpiI-BpiI-DR-EF1 ⁇ t-mCherry were generated to knockdown target genes by transient transfection.
  • the gRNA oligos were annealed and ligated into BpiI sites.
  • the gRNA sequences were listed below.
  • HEK293T cell lines were purchased from Stem Cell Bank, Chinese Academy of Sciences. HEK293T cell lines were cultured with DMEM (Gibco) supplemented with 10%fetal bovine serum (Gibco) , 1%penicillin/streptomycin (Thermo Fisher Scientific) and 0.1 mM non-essential amino acids (Gibco) in an incubator at 37°C with 5%CO 2 . When cells reached 90%confluence, HEK293T cells were passaged at a ratio of 1 ⁇ 4 to 12-well plates. After 12 hr, 2 ⁇ g/well plasmids were transfected into cells with Lipofectamine 3000 (Thermo Fisher Scientific) using the standard protocol.
  • RNA extraction 48 hr after transfection, 50,000 of both EGFP and mCherry positive cells were sorted by BD FACS Aria II for RNA extraction.
  • mCherry knockdown total cells of the 12-well plate were collected for RNA extraction.
  • Flow cytometry results were analyzed with FlowJo V10.5.3.
  • transgene cell lines cells were expanded cultivation for dox (1 ⁇ g/mL) induction.
  • qPCR reactions were performed with AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech) . All of the reagents were precooled in advance. qPCR results were analyzed with - ⁇ Ct method.
  • I-TASSER were used to perform the protein structure prediction.
  • Cas13 protein purification was performed according to protocol as previously described.
  • the humanized codon-optimized gene for Cas13d/cfCas13d/Cas13e/cfCas13e was synthesized (Huagene) and cloned into a bacterial expression vector (pC013-Twinstrep-SUMO-huLwCas13a, Plasmid #90097) after the plasmid digestion by BamHI and NotI with NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) .
  • the expression constructs were transformed into BL21 (DE3) (TIANGEN) cells.
  • BL21 DE3 (TIANGEN) cells.
  • LB Broth growth media Teryptone 10.0 g; Yeast Extract 5.0 g; NaCl 10.0 g, Sangon Biotech
  • Cells were then grown to a cell density A600 of 0.6 at 37°C, and then SUMO-Cas13 proteins expression was induced by supplementing with 500 mM IPTG.
  • the induced cells were grown at 16°C for 16-18 hours before harvest by centrifuge (4,000 rpm, 20 min) . Collected cells were resuspended in Buffer W (Strep-Tactin Purification Buffer Set, IBA) and lysed using ultrasonic homogenizer (Scientz) .
  • Buffer W Stringep-Tactin Purification Buffer Set, IBA
  • ultrasonic homogenizer Scientz
  • Fluorescent labeled ssRNA reporter assay for Cas13 nuclease activity was performed as previously described. For on-target cleavage activity analysis, assays were performed with 45 nM purified Cas13d/cfCas13d/Cas13e/cfCas13e, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (Sangon Biotech) , 1 ⁇ L murine RNase inhibitor (New England Biolabs) , 100 ng of background total human RNA (purified from HEK293T cell culture) , and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl including 25mM Tris-HCl, pH7.5 and 25mM Tris-HCl, pH7.0, 60 mM NaCl, 6 mM MgCl 2 , pH 7.3) . Reactions were allowed to proceed for 1-3 hr at 37°C on
  • RNA-seq library was generated and quality was assessed using Illumina Hiseq X-ten platform in Novogene.
  • Single cell clones with dCas13d /Cas13d /cfCas13d and RPL4 gRNA were plated on a 24-well plate at 2 ⁇ 10 5 cells/mL with or without dox treated (1 ⁇ g/mL) . Cell were collected at 24, 48, 72, 96 and 120 hrs. Cell number was counted by an automated cell counter (C10311, Invitrogen) . Experiments were performed for three replicates.
  • Cell proliferation was assessed by using a colorimetric thiazolyl blue (MTT) assay. Briefly, single cell clones with dCas13d /Cas13d /cfCas13d and RPL4 gRNA were treated with or without dox treated (1 ⁇ g/mL) for 0, 24, 48, 72, 96 or 120 hrs. Then each group of cells was collected and further plated on a 24-well plate at 2 ⁇ 10 5 cells/mL with or without dox treated (1 ⁇ g/mL) .
  • MTT colorimetric thiazolyl blue
  • the tetrazolium salt MTT (Sigma-Chemie) was added to a final concentration of 2 ⁇ g/mL, and incubation was continued for 4 hrs. Cells were washed 3 times and finally lysed with dimethyl sulfoxide. Metabolization of MTT directly correlates with the cell number and was quantitated by measuring the absorbance at 550 nm (reference wavelength, 690 nm) by using a microplate reader (type 7500; Cambridge Technology, Watertown, MA) . Experiments were performed for five replicates.

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Abstract

L'invention concerne de nouvelles enzymes effectrices CRISPR/Cas modifiées, telles que Cas13 (par exemple Cas13d et Cas13e) maintenant particulièrement l'activité endonucléase spécifique de la séquence guide et ne présentant pas particulièrement d'activité endonucléase collatérale indépendante de la séquence guide par rapport à la Cas de type sauvage correspondante. L'invention concerne également des polynucléotides codant lesdites enzymes, des vecteurs ou des cellules hôtes comprenant les polynucléotides ou les Cas modifiées, et un procédé d'utilisation, tel que dans le knock down de transcription de gènes cibles basé sur l'ARN.
PCT/CN2021/079821 2020-09-30 2021-03-09 Système crispr/cas13 modifié et ses utilisations Ceased WO2022188039A1 (fr)

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PCT/CN2021/079821 WO2022188039A1 (fr) 2021-03-09 2021-03-09 Système crispr/cas13 modifié et ses utilisations
PCT/CN2021/121926 WO2022068912A1 (fr) 2020-09-30 2021-09-29 Système crispr/cas13 modifié et ses utilisations
CN202180018124.0A CN116096875B (zh) 2020-09-30 2021-09-29 工程化的CRISPR/Cas13系统及其用途
CN202410088378.5A CN118109438A (zh) 2020-09-30 2021-09-29 工程化的CRISPR/Cas13系统及其用途
EP21793851.3A EP4222253A1 (fr) 2020-09-30 2021-09-29 Système crispr/cas13 modifié et ses utilisations
EP22710479.1A EP4305157A1 (fr) 2021-03-09 2022-03-09 Système crispr/cas13 ingéniérisé et ses utilisations
CN202280003194.3A CN115427561B (zh) 2021-03-09 2022-03-09 工程化CRISPR/Cas13系统及其用途
PCT/CN2022/079890 WO2022188797A1 (fr) 2021-03-09 2022-03-09 Système crispr/cas13 ingéniérisé et ses utilisations
US17/836,175 US20220389398A1 (en) 2020-09-30 2022-06-09 Engineered crispr/cas13 system and uses thereof
US17/836,266 US20230075045A1 (en) 2021-03-09 2022-06-09 Engineered crispr/cas13 system and uses thereof

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WO2023069987A1 (fr) 2021-10-20 2023-04-27 University Of Rochester Traitement de régénération de référence croisée de perte de matière blanche liée à l'âge à une application associée
WO2024149283A1 (fr) * 2023-01-10 2024-07-18 上海交通大学 Mutant présentant une activité casrx et son utilisation
WO2025090427A1 (fr) 2023-10-23 2025-05-01 University Of Rochester Soulagement d'hyperexcitabilité ciblé glial dans des maladies neurodégénératives
WO2025095255A1 (fr) * 2023-11-03 2025-05-08 서울대학교산학협력단 Procédé de régulation de traduction dans des bactéries par utilisation de cas13d morte

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023069987A1 (fr) 2021-10-20 2023-04-27 University Of Rochester Traitement de régénération de référence croisée de perte de matière blanche liée à l'âge à une application associée
WO2023069979A1 (fr) 2021-10-20 2023-04-27 University Of Rochester Cellules progénitrices gliales isolées destinées à être utilisées dans le traitement par compétition de la perte de matière blanche liée à l'âge
WO2024149283A1 (fr) * 2023-01-10 2024-07-18 上海交通大学 Mutant présentant une activité casrx et son utilisation
WO2025090427A1 (fr) 2023-10-23 2025-05-01 University Of Rochester Soulagement d'hyperexcitabilité ciblé glial dans des maladies neurodégénératives
WO2025095255A1 (fr) * 2023-11-03 2025-05-08 서울대학교산학협력단 Procédé de régulation de traduction dans des bactéries par utilisation de cas13d morte

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