Type vi-e and type vi-f crispr-cas system and uses thereof

Abstract

To provide novel CRISPR / Cas compositions and uses thereof for targeting nucleic acids.SOLUTION: The invention provides non-naturally occurring or engineered RNA-targeting systems, comprising a novel RNA-targeting Cas13e or Cas13f effector protein, and at least one targeting nucleic acid component, such as a guide RNA (gRNA) or crRNA. The novel Cas effector protein is one of the smallest known Cas effector proteins, at about 800 amino acids in size, and are thus uniquely suitable for delivery using vectors of small capacity, such as an AAV vector.SELECTED DRAWING: None

Description

[Background technology]

[0001] CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotes, such as bacteria and archaea. The 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 from similar bacteriophages during subsequent infection of the prokaryote.

[0002] The CRISPR-associated system is a set of homologous genes, or Cas genes, some of which encode Cas proteins with helicase and nuclease activity. Cas proteins are enzymes that use the RNA derived from the CRISPR sequence (crRNA) as a guide sequence to recognize and cleave specific strands of polynucleotides (e.g., DNA) that are complementary to the crRNA.

[0003] Together, the CRISPR-Cas systems constitute a primitive prokaryotic "immune system" that confers resistance or acquired immunity to foreign pathogenic genetic elements, such as genetic elements present in extrachromosomal DNA (e.g., plasmids) and bacteriophages, or foreign RNA encoded by the foreign DNA.

[0004] In nature, the CRISPR / Cas system appears to be a widespread prokaryotic defense mechanism against foreign genetic material, being found in roughly 50% of sequenced bacterial genomes and nearly 90% of sequenced archaeal genomes. This prokaryotic system has since been developed to form the basis of a technology known as CRISPR-Cas that has found widespread use in many eukaryotic organisms, including humans, in a wide variety of applications, including basic biological research, development of biotechnology products, and disease treatment.

[0005] Prokaryotic CRISPR-Cas systems contain an extremely diverse collection of protein effectors, non-coding elements, and locus architectures, some examples of which have been engineered and configured to yield important biotechnological results.

[0006] CRISPR locus structure has been studied in many systems. In these systems, CRISPR arrays in genomic DNA typically contain short DR sequences separated by unique spacer sequences following an AT-rich leader sequence. The CRISPR DR sequences typically range in size from 28 to 37 bp, but can range from 23 to 55 bp. Some DR sequences exhibit dyad symmetry, implying the formation of secondary structures such as stem-loops ("hairpins") in RNA. Others appear to be unstructured. The size of the spacer in various CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). Usually, there are less than 50 units of repeat-spacer sequences in a CRISPR array.

[0007] Small clusters of cas genes are often found next to such CRISPR repeat-spacer arrays. To date, the 93 cas genes identified have been classified into 35 families based on the sequence similarity of the encoded proteins. Eleven of the 35 families form the so-called cas core, which includes the protein families Cas1 to Cas9. A complete CRISPR-Cas locus is a cas core. It has at least one gene belonging to A.

[0008] 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. The single-subunit effector compositions of class 2 systems provide a simpler set of components for engineering and application translation and so far have been an important source of discovery, engineering, and optimization of novel, powerful, programmable technologies for genome engineering and beyond.

[0009] Class 1 systems are further divided into types I, III, and IV; class 2 systems are divided into types II, V, and VI. These six system types are further divided into 19 subtypes. The classification is also based on the complement of cas genes present. Most CRISPR-Cas systems have a Cas1 protein. Many prokaryotes contain multiple CRISPR-Cas systems. This suggests that multiple CRISPR-Cas systems may be compatible and share components.

[0010] One of the first and best characterized Cas proteins, Cas9, is a typical member of class 2, type II, and is derived from Streptococcus pyogenes (SpCas9). Cas9 is a DNA endonuclease that is activated by a small crRNA molecule that complements the target DNA sequence and a separate transactivating CRISPR RNA (tracrRNA). The crRNA consists of a direct repeat (DR) sequence that carries the protein that binds to the crRNA, and a spacer sequence, which can be engineered to be complementary to any desired nucleic acid target sequence. In this way, the CRISPR system can be programmed to target DNA or RNA targets by modifying the spacer sequence of the crRNA. The crRNA and tracrRNA have been fused to form a single guide RNA (sgRNA) for better practical utility. When combined with Cas9, the sgRNA hybridizes to its target DNA and guides Cas9 to cleave the target DNA. Additionally, other Cas9 effector proteins from other species have been identified and used as well, including the Cas9 from the S. thermophilus CRISPR system. These 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.

[0011] Another recently characterized Cas effector protein is Cas12a (previously known as Cpf1). Cas12a, along with C2c1 and C2c3, is a member of the class 2, type V Cas proteins that lack HNH nuclease but possess RuvC nuclease activity. Cas12a was first characterized in the CRISPR / Cpf1 system of the bacterium Francisella novicida. Its original name reflects the prevalence of its CRISPR-Cas subtype in Prevotella and Francisella lineages. Cas12a has been compared to Cas9 It exhibits several key differences: it induces "staggered" cuts in double-stranded DNA as opposed to the "blunt" cuts made by Cas9, it relies on a "T-rich" PAM sequence (providing an alternative targeting site for Cas9), and it requires only CRISPR RNA (crRNA) and not tracrRNA for successful targeting. Cas12a's small crRNA is better suited than Cas9 for multiplex genome editing because more of it can be packaged into a vector than Cas9's sgRNA. Furthermore, the sticky 5' overhangs left by Cas12a can be used for DNA assembly that is much more target-specific than traditional restriction enzyme cloning. Finally, Cas12a cleaves DNA 18-23 base pairs downstream from its PAM site. This means that after DNA repair by the NHEJ system after the creation of double-strand breaks (DSB), Cas12a allows multiple rounds of DNA cleavage, as opposed to the possible one round after Cas9 cleavage, due to the absence of interruptions in the nuclease recognition sequence.Because the Cas9 cleavage sequence is only three base pairs upstream of the PAM site, the NHEJ pathway typically prevents further rounds of cleavage by generating indel mutations that destroy the recognition sequence.Theoretically, repeated rounds of DNA cleavage are associated with an increased probability of genome editing that is desired to occur.

[0012] More recently, several class 2, type VI Cas proteins have been identified, including Cas13 (also known as C2c2), Cas13b, Cas13c, and Cas13d. Each is an RNA-guided RNase (i.e., these Cas proteins recognize target RNA sequences rather than target DNA sequences using crRNAs, as in Cas9 and Cas12a). Overall, the CRISPR / Cas13 system can achieve higher RNA digestion efficiency compared to traditional RNAi and CRISPRi technologies, while simultaneously exhibiting significantly less off-target cleavage compared to RNAi.

[0013] One drawback of these currently identified Cas13 proteins is their relatively large size. Cas13a, Cas13b, and Cas13c each have more than 1100 amino acid residues. Therefore, if at all possible, it is difficult to package the coding sequence (about 3.3 kb) and sgRNA, plus any required promoter and translational regulatory sequences, into a certain small capacity gene therapy vector, for example, the most effective and safest gene therapy vector currently based on adeno-associated virus (AAV), which has a packaging capacity of about 4.7 kb. Only Cas13d, the smallest Cas13 protein to date, has about 920 amino acids (i.e., about 2.8 kb of coding sequence) and can theoretically be packaged into AAV vectors, but its use is limited to single base editing-based gene therapy, which relies on using Cas13d-based fusion proteins with single base editing function, such as dCas13d-ADAR2DD (having about 3.9 kb of coding sequence).

[0014] Furthermore, all currently known Cas13 proteins / systems have non-specific / collateral RNase activity immediately after activation by crRNA-based target sequence recognition. This activity is particularly strong in Cas13a and Cas13b, and is still detectably present in Cas13d. While this property can be advantageously used in nucleic acid detection methods, the non-specific / collateral RNase activity of these Cas13 proteins constitutes a considerable potential hazard for gene therapy uses. Summary of the Invention [Means for solving the problem]

[0015] One aspect of the present invention is a method for producing a nucleic acid sequence comprising: (1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA and a direct repeat (DR) sequence on the 3' side of the spacer sequence; and (2) a CRI having any one of the amino acid sequences of SEQ ID NOs: 1 to 7. The present invention provides a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex comprising an SPR-associated protein (Cas), or a derivative or functional fragment of said Cas; wherein the Cas, said derivatives and functional fragments are capable of (i) binding to an RNA guide sequence, and (ii) targeting a target RNA, provided that the spacer sequence is not 100% complementary to a naturally occurring bacteriophage nucleic acid, where the complex comprises a Cas of any one of SEQ ID NOs: 1-7, or the target RNA is encoded by eukaryotic DNA.

[0016] In certain embodiments, the DR sequence has substantially the same secondary structure as that of any one of SEQ ID NOs:8-14.

[0017] In certain embodiments, the DR sequence is encoded by any one of SEQ ID NOs:8-14.

[0018] In certain embodiments, the target RNA is encoded by eukaryotic DNA.

[0019] In certain embodiments, the eukaryotic DNA is non-human mammalian DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, avian DNA, reptile DNA, rodent DNA, fish DNA, worm / nematode DNA, yeast DNA.

[0020] In certain embodiments, the target RNA is mRNA.

[0021] In certain embodiments, the spacer sequence is between 15 and 55 nucleotides, between 25 and 35 nucleotides, or about 30 nucleotides.

[0022] In certain embodiments, the spacer sequence is 90-100% complementary to the target RNA.

[0023] In certain embodiments, the derivatives comprise conservative amino acid substitutions of one or more residues of any one of SEQ ID NOs:1-7.

[0024] In certain embodiments, the derivatives contain only conservative amino acid substitutions.

[0025] In certain embodiments, the derivative has a sequence identical to a wild-type Cas of any one of SEQ ID NOs: 1-7 within the HEPN domain or RXXXXH motif.

[0026] In certain embodiments, the derivatives are capable of binding to an RNA guide sequence that hybridizes to a target RNA, but do not have RNase catalytic activity due to a mutation in the RNase catalytic site of Cas.

[0027] In certain embodiments, the derivative has an N-terminal deletion of up to 210 residues and / or a C-terminal deletion of up to 180 residues.

[0028] In certain embodiments, the derivative has an N-terminal deletion of about 180 residues and / or a C-terminal deletion of about 150 residues.

[0029] In certain embodiments, the derivative further comprises an RNA base-editing domain.

[0030] In certain embodiments, the RNA base-editing domain is an adenosine deaminase, such as a double-stranded RNA-specific adenosine deaminase (e.g., ADAR1 or ADAR2). apolipoprotein B mRNA editing enzyme; catalytic polypeptide-like (APOBEC); or activation-induced cytidine deaminase (AID).

[0031] In certain embodiments, the ADAR has an E488Q / T375G double mutation or is ADAR2DD.

[0032] In certain embodiments, the base-editing domain is further fused to an RNA binding domain, such as MS2.

[0033] In certain embodiments, the derivative further comprises an RNA methyltransferase, an RNA demethylase, an RNA splicing modifier, a localization factor, or a translation modifier.

[0034] In certain embodiments, the Cas, derivative, or functional fragment comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).

[0035] In certain embodiments, targeting of the target RNA results in modification of the target RNA.

[0036] In certain embodiments, the modification of the target RNA is cleavage of the target RNA.

[0037] In certain embodiments, the modification of the target RNA is the deamination of adenosine (A) to inosine (I).

[0038] In certain embodiments, the CRISPR-Cas complex of the invention further comprises a target RNA comprising a sequence capable of hybridizing to the spacer sequence.

[0039] Another aspect of the present invention provides a fusion protein comprising (1) a Cas of the present invention, a derivative thereof, or a functional fragment thereof, and (2) a heterologous functional domain.

[0040] In certain embodiments, the heterologous functional domain is: a nuclear localization signal (NLS), a reporter protein or detection label (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), a localization signal, a protein targeting moiety, a DNA binding domain (e.g., MBP, Lex A DBD, Gal4 DBD), epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc.), transcription activation domain (e.g., VP64 or VPR), transcription inhibitory domain (e.g., a KRAB portion or a SID portion), nuclease (e.g., FokI), deamination domain (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methylase, demethylase, transcription release factor, HDAC, polypeptide having ssRNA cleavage activity, polypeptide having dsRNA cleavage activity, polypeptide having ssDNA cleavage activity, polypeptide having dsDNA cleavage activity, DNA or RNA ligase, or any combination thereof.

[0041] In certain embodiments, the heterologous functional domain is fused N-terminally, C-terminally, or internally within the fusion protein.

[0042] Another aspect of the present invention provides a conjugate comprising (1) a Cas of the present invention, a derivative thereof, or a functional fragment thereof, conjugated to (2) a heterologous functional moiety.

[0043] In certain embodiments, the heterologous functional moiety is: a nuclear localization signal (NLS), a reporter protein, or a detectable label (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), localization signals, 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 inhibitory domains (e.g., a KRAB moiety or a SID moiety), nucleases (e.g., FokI), deamination domains (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methylases, demethylases, transcription release factors, HDACs, polypeptides having ssRNA cleavage activity, polypeptides having dsRNA cleavage activity, polypeptides having ssDNA cleavage activity, polypeptides having dsDNA cleavage activity, DNA or RNA ligases, or any combination thereof.

[0044] In certain embodiments, the heterologous functional moiety is conjugated N-terminally, C-terminally, or internally to the Cas, a derivative, or a functional fragment thereof.

[0045] Another aspect of the present invention provides a polynucleotide encoding any one of SEQ ID NOs: 1 to 7, or a derivative thereof, or a functional fragment thereof, or a fusion protein thereof, provided that the polynucleotide is not any one of SEQ ID NOs: 15 to 21.

[0046] In certain embodiments, the polynucleotide is codon optimized for expression in a cell.

[0047] In certain embodiments, the cell is a eukaryotic cell.

[0048] Another aspect of the present invention provides a non-naturally occurring polynucleotide comprising a derivative of any one of SEQ ID NOs:8-14, the derivative (i) having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotide additions, deletions, or substitutions compared to any one of SEQ ID NOs:8-14; (ii) having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 97% sequence identity to any one of SEQ ID NOs:8-14; (iii) hybridizing under stringent conditions to any one of SEQ ID NOs:8-14, or to any of (i) and (ii); or (iv) being the complement of any of (i)-(iii), provided that the derivative is not any of SEQ ID NOs:8-14, and the derivative encodes (or is) an RNA that maintains substantially the same secondary structure as any of the RNAs encoded by SEQ ID NOs:8-14.

[0049] In certain embodiments, the derivative functions as a DR sequence of any one of the Cas of the invention, its derivatives, or functional fragments thereof.

[0050] Another aspect of the present invention provides a vector comprising a polynucleotide of the present invention.

[0051] In certain embodiments, the polynucleotide is operably linked to a promoter and, optionally, an enhancer.

[0052] In certain embodiments, the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue-specific promoter.

[0053] In certain embodiments, the vector is a plasmid.

[0054] In certain embodiments, the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an AAV vector, Or a lentiviral vector.

[0055] In certain embodiments, the AAV vector is a recombinant AAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0056] Another aspect of the present invention provides a delivery system comprising: (1) a delivery vehicle; and (2) a CRISPR-Cas complex of the present invention, a fusion protein of the present invention, a conjugate of the present invention, a polynucleotide of the present invention, or a vector of the present invention.

[0057] In certain embodiments, the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0058] Another aspect of the present invention provides a cell or its progeny comprising a CRISPR-Cas complex of the present invention, a fusion protein of the present invention, a conjugate of the present invention, a polynucleotide of the present invention, or a vector of the present invention.

[0059] In certain embodiments, the cell or its progeny is a eukaryotic cell (eg, a non-human mammalian cell, a human cell, or a plant cell) or a prokaryotic cell (eg, a bacterial cell).

[0060] Another aspect of the invention provides a non-human multicellular eukaryotic organism comprising a cell of the invention.

[0061] In certain embodiments, the non-human multicellular eukaryote is an animal (eg, rodent or primate) model for a human genetic disorder.

[0062] Another aspect of the invention provides a method of modifying a target RNA, the method comprising contacting the target RNA with a CRISPR-Cas complex of the invention, wherein a spacer sequence is complementary to at least 15 nucleotides of the target RNA; the Cas, derivative, or functional fragment binds to an RNA guide sequence to form a complex; the complex binds to the target RNA; and upon binding of the complex to the target RNA, the Cas, derivative, or functional fragment modifies the target RNA.

[0063] In certain embodiments, the target RNA is modified by cleavage by Cas.

[0064] In certain embodiments, the target RNA is modified by deamination with a derivative comprising a double-stranded RNA-specific adenosine deaminase.

[0065] In certain embodiments, the target RNA is mRNA, tRNA, rRNA, non-coding RNA, lncRNA, or nuclear RNA.

[0066] In certain embodiments, upon binding of the complex to the target RNA, the Cas, derivatives, and functional fragments do not exhibit substantial (or detectable) collateral RNase activity.

[0067] In certain embodiments, the target RNA is intracellular.

[0068] In certain embodiments, the cell is a cancer cell.

[0069] In certain embodiments, the cells are infected with an infectious agent.

[0070] In certain embodiments, the infectious agent is a virus, a prion, a protozoan, a fungus, or a parasite. It's an insect.

[0071] In certain embodiments, the CRISPR-Cas complex is encoded by a first polynucleotide encoding any one of SEQ ID NOs: 1-7, or a derivative or functional fragment thereof, and a second polynucleotide comprising any one of SEQ ID NOs: 8-14, and a sequence encoding a spacer RNA capable of binding to a target RNA, and the first and second polynucleotides are introduced into a cell.

[0072] In certain embodiments, the first and second polynucleotides are introduced into the cell by the same vector.

[0073] In certain embodiments, the method causes one or more of the following: (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 proliferation 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.

[0074] Another aspect of the invention provides a method of treating a condition or disease in a subject in need of such treatment, the method comprising administering to the subject a composition comprising a CRISPR-Cas complex of the invention, or a polynucleotide encoding same; the spacer sequence is complementary to at least 15 nucleotides of a target RNA associated with the condition or disease; the Cas, derivative, or functional fragment binds to the RNA guide sequence to form a complex; the complex binds to the target RNA; and upon binding of the complex to the target RNA, the Cas, derivative, or functional fragment cleaves the target RNA, thereby treating the condition or disease in the subject.

[0075] In certain embodiments, the condition or disease is cancer or an infectious disease.

[0076] In certain embodiments, the cancer is Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, uterine cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or bladder cancer.

[0077] In certain embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method.

[0078] Another aspect of the invention provides a cell or its progeny obtained by the method of the invention, wherein the cell and its progeny comprise a non-naturally occurring modification (e.g., a non-naturally occurring modification in the transcribed RNA of the cell / progeny).

[0079] Another aspect of the invention provides a method for detecting the presence of a target RNA, the method comprising contacting the target RNA with a composition comprising a fusion protein of the invention, or a conjugate of the invention, or a polynucleotide encoding the fusion protein, wherein the fusion protein or conjugate comprises a detectable label (e.g., one that can be detected by fluorescence, Northern blot, or FISH) and a synthetic spacer sequence capable of binding to the target RNA.

[0080] Another aspect of the invention provides a eukaryotic cell comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex, the CRISPR-Cas complex comprising: ) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA, and a direct repeat (DR) sequence 3' to the spacer sequence; and (2) a CRISPR-associated protein (Cas) having an amino acid sequence of any one of SEQ ID NOs: 1 to 7, or a derivative or functional fragment of said Cas; wherein the Cas, derivative and functional fragment are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.

[0081] It is to be understood that any embodiment of the invention described herein, including those described only in the examples or claims, or only in one aspect / section below, can be combined with any other one or more embodiments of the invention, unless expressly denied or inappropriate. [Brief description of the drawings]

[0082] [Figure 1] Figure 1 is a schematic (not to scale) representation of the genomic loci of representative Cas13e and Cas13f family members. The Cas coding sequence (long bar with pointed ends) is followed by multiple nearby direct repeats (DRs) (shorter bars) and spacer sequences (diamonds). [Diagram 2] Figure 2 shows the predicted secondary structures of the DR sequences associated with each Cas13e and Cas13f protein, whose coding sequences are represented, from left to right, by SEQ ID NOs: 8-14, respectively. [Diagram 3] FIG. 3 shows a phylogenetic tree for the newly discovered Cas13e and Cas13f effector proteins of the present invention, as well as the related previously discovered Cas13a, Cas13b, Cas13c, and Cas13d effector proteins. [Figure 4] Figure 4 shows the domain structure for the Cas13a-Cas13f proteins, showing the overall size of each representative member of the Cas proteins and the location of the two RXXXXH motifs. [Diagram 5]FIG. 5 shows the predicted 3D structure of the Cas13e.1 effector protein. [Figure 6] FIG. 6 is a schematic diagram showing that three plasmids encoding (1) a Cas13e effector protein, (2) a coding sequence for a guide RNA (gRNA) that is complementary to mCherry mRNA and can generate a guide RNA that can form a complex with the Cas13e effector protein, and (3) an mCherry reporter gene, can be transfected into cells to express each gene product, resulting in degradation of the reporter mCherry mRNA. [Figure 7] Figure 7 shows the knockdown of mCherry mRNA by a guide RNA complementary to mCherry mRNA, as evidenced by the reduction of mCherry expression under a fluorescent microscope. As a negative control, a non-targeting (NT) guide RNA that does not hybridize / bind to mCherry mRNA failed to knockdown mCherry expression. [Figure 8] FIG. 8 shows approximately 75% knockdown of mCherry expression in the experiment of FIG. [Figure 9] FIG. 9 shows that Cas13e utilizes guide RNAs with DR sequences at the 3′ end (as opposed to DR sequences at the 5′ end of the guide RNA). [Figure 10] FIG. 10 shows the correlation between spacer sequence length and target RNA-specific (guide RNA-dependent) RNase activity compared to non-targeting (NT) controls. [Figure 11] FIG. 11 shows the correlation between spacer sequence length and non-specific / collateral (guide RNA-independent) RNase activity against target RNA compared to non-targeting (NT) controls. [Figure 12]Figure 12 shows that the dCas13e.1-ADAR2DD fusion has RNA base editing activity. Specifically, three plasmids each encoding (1) dCas13e (RNase dead) protein fused to single base RNA editor ADAR2DD, (2) a coding sequence of a guide RNA (gRNA) that can generate a guide RNA that is complementary to mutant mCherry mRNA with a point mutation from G to A and can form a complex with dCas13e effector protein, and (3) a mutant mCherry reporter gene that encodes mCherry mRNA with a point mutation from G to A can be transfected into cells to express their respective gene products. Mutant mCherry mRNA is usually unable to generate fluorescent mCherry protein due to point mutation. When the guide RNA binds to mutant mCherry mRNA, the fused ADAR2DD base editor converts A to I (G equivalent), thus restoring the ability of the mRNA to code for fluorescent mCherry protein. [Figure 13] Figure 13 shows the restoration of mCherry expression as a result of successful RNA base editing. In the experiment of Figure 12, the plasmid encoding mutant mCherry (mCherry*) alone did not express fluorescent mCherry. The plasmid encoding the dCas13e-ADAR2DD base editor alone also did not express fluorescent mCherry. The plasmid encoding either gRNA-1 or gRNA-2 alone (which also express a GFP reporter) also did not express fluorescent mCherry, but GFP was prominently expressed. However, significant fluorescent mCherry expression (along with GFP reporter expression) was observed when all three plasmids were transfected into the same cells. [Figure 14] FIG. 14 shows the relevant segment of a mutant mCherry gene with a premature stop codon TAG, the sequences of two gRNAs that can be complexed with the dCas13e-ADAR2DD RNA base editor, and the "corrected" TGG codon. [Figure 15]FIG. 15 is a schematic (not to scale) showing a series of progressive C-terminal deletion constructs of dCas13e.1 fused to the ADAR2DD RNA base editor (denoted as "ADAR2") and other transcriptional regulatory elements. [Figure 16] FIG. 16 shows the percentage of mCherry mutant conversion results back to wild-type mCherry for the series of C-terminal deletion mutants in FIG. [Figure 17] FIG. 17 is a schematic (not to scale) showing a series of progressive C-terminal and, optionally, N-terminal deletion constructs of dCas13e.1 fused to the ADAR2DD RNA base editor. [Figure 18] FIG. 18 shows the percentage of mCherry mutant conversion results back to wild-type mCherry for selected C- and N-terminal deletion mutants in FIG. [Figure 19] FIG. 19 shows a series of plasmids encoding Cas13a, Cas13b, Cas13d, Cas13e.1, and Cas13f.1, an mCherry reporter gene, and an ANXA4-targeting gRNA coding sequence or a non-targeting gRNA as a control. [Figure 20] FIG. 20 shows effective knockdown of ANXA4 expression by Cas13e.1, Cas13f.1, Cas13a, and Cas13d. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] 1. Overview The invention described herein provides novel class 2, type VI Cas effector proteins, sometimes referred to herein as Cas13e and Cas13f. The novel Cas13 proteins of the invention are much smaller than previously discovered Cas13 effector proteins (Cas13a-Cas13d) such that they can be easily packaged into low-volume gene therapy vectors, such as AAV vectors, along with their crRNA coding sequences. Furthermore, compared to Cas13a, Cas13b, and Cas13d effector proteins, the newly discovered Cas13e and Cas13f effector proteins are more potent in knocking down RNA target sequences and are more efficient ... While being more efficient in single base editing, they exhibit negligible nonspecific / collateral RNase activity after activation by crRNA-based target recognition, except when the spacer sequence is within a specific narrow range (e.g., about 30 nucleotides), making these new Cas proteins ideally suited for gene therapy.

[0084] Thus, in a first aspect, the present invention provides Cas13e and Cas13f effector proteins, such as those having the amino acid sequences of SEQ ID NOs: 1-7, or their orthologues, homologues, various derivatives (described herein below), functional fragments (described herein below), wherein said orthologues, homologues, derivatives and functional fragments maintain at least one function of any one of the proteins of SEQ ID NOs: 1-7. Such functions include, but are not limited to, the ability to bind to and form a complex with a guide RNA / crRNA (described herein below) of the present invention, RNase activity, and the ability to bind to and cleave a target RNA at a specific site under the guidance of a crRNA that is at least partially complementary to the target RNA.

[0085] In certain embodiments, the Cas13e or Cas13f effector protein of the present invention may be (i) any one of SEQ ID NOs: 1-7; (ii) a derivative having one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues) of any one of SEQ ID NOs: 1-7, including additions, deletions, and / or substitutions (e.g., conserved substitutions); or (iii) a derivative having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to any one of SEQ ID NOs: 1-7.

[0086] In certain embodiments, the Cas13e and Cas13f effector proteins, orthologs, homologs, derivatives and functional fragments thereof are not naturally occurring, e.g., have at least one amino acid difference compared to a naturally occurring sequence.

[0087] In a related aspect, the present invention provides further derivative Cas13e and Cas13f effector proteins based on any one of SEQ ID NOs: 1-7, or the above orthologues, homologues, derivatives and functional fragments thereof, comprising another covalently or non-covalently linked protein or polypeptide or other molecule (such as a detection reagent or drug / chemical moiety). Such other protein / polypeptide / other molecule may be linked, for example, via chemical bonds, genetic fusion or other non-covalent bonds (such as biotin-streptavidin bonds). Such derivative proteins do not affect the functions of the original proteins, for example, their ability to bind and form a complex with the guide RNA / crRNA of the present invention (described later in this specification), their RNase activity and their ability to bind and cleave the target RNA at a specific site under the guidance of a crRNA that is at least partially complementary to the target RNA.

[0088] Such induction can be used, for example, to add a nuclear localization signal (such as an NLS, such as the SV40 large T antigen NLS) to enhance the ability of the Cas13e and Cas13f effector proteins of the present invention to enter the cell nucleus. Such induction can also be used to add a targeting molecule or moiety to direct the Cas13e and Cas13f effector proteins of the present invention to a specific cell or subcellular location. Such induction can also be used to add a detectable label to facilitate detection, monitoring, or purification of the Cas13e and Cas13f effector proteins of the present invention. Furthermore, such induction can be used to add a deamination enzyme moiety (such as one with adenine or cytosine deamination activity) to facilitate RNA base editing.

[0089] The induction may be achieved by ligating the N-terminus or ligation of the Cas13e and Cas13f effector proteins of the present invention. This can be done by adding the further moiety either at the C-terminus or internally (eg, by internal fusion or attachment via the side chain of an internal amino acid).

[0090] In a related second aspect, the invention provides conjugates of the Cas13e and Cas13f effector proteins of the invention based on any one of SEQ ID NOs: 1-7, or the above orthologues, homologues, derivatives and functional fragments thereof, which are conjugated to a moiety such as another protein or polypeptide, a detectable label, or a combination thereof. Such conjugated moieties include, but are not limited to, localization signals, reporter genes (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), labels (e.g., fluorescent dyes such as FITC or DAPI), NLS, targeting moieties, DNA binding domains (e.g., MBP, Lex A DBD, Gal4, Glutathione ... DBD), epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc.), transcription activation domain (e.g., VP64 or VPR), transcription inhibitory domain (e.g., a KRAB portion or a SID portion), nuclease (e.g., FokI), deamination domain (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methylase, demethylase, transcription release factor, HDAC, ssRNA cleavage activity, dsRNA cleavage activity, ssDNA cleavage activity, dsDNA cleavage activity, DNA or RNA ligase, any combination thereof, and the like.

[0091] For example, the conjugate may include one or more NLSs, which may be located at or near the N-terminus, the C-terminus, internally, or combinations thereof. Conjugation may be via an amino acid (such as D or E, or S or T), an amino acid derivative (such as Ahx, β-Ala, GABA or Ava), or a PEG linkage.

[0092] In certain embodiments, the conjugation does not affect the functions of the original protein, such as its ability to bind and form a complex with the guide RNA / crRNA of the invention (described later in this specification), its RNase activity, and its ability to bind and cleave the target RNA at a specific site under the guidance of a crRNA that is at least partially complementary to the target RNA.

[0093] In a related third aspect, the present invention provides fusions of the Cas13e and Cas13f effector proteins of the invention based on any one of SEQ ID NOs: 1 to 7, or the above-mentioned orthologues, homologues, derivatives and functional fragments thereof, which fusions can include a localization signal, a reporter gene (e.g. GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), a NLS, a protein targeting moiety, a DNA binding domain (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 inhibitory domains (e.g., KRAB or SID portions), nucleases (e.g., FokI), deamination domains (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methylases, demethylases, transcription release factors, HDACs, ssRNA cleavage activity, dsRNA cleavage activity, ssDNA cleavage activity, dsDNA cleavage activity, DNA or RNA ligase, or any combination thereof.

[0094] For example, the fusion may include one or more NLSs, which may be located at or near the N-terminus, C-terminus, internally, or a combination thereof. In certain embodiments, the conjugation does not affect the functions of the original protein, such as its ability to bind and form a complex with the guide RNA / crRNA of the invention (described later herein), its RNase activity, and its ability to bind and cleave the target RNA at a specific site under the guidance of a crRNA that is at least partially complementary to the target RNA.

[0095] In a fourth aspect, the present invention provides an isolated polynucleotide comprising: (i) any one of SEQ ID NOs: 8 to 14; (ii) a polynucleotide having one, two, three, four, or five nucleotides deleted, added, and / or substituted compared to any one of SEQ ID NOs: 8 to 14; (iii) a polynucleotide sharing at least 80%, 85%, 90%, 95% sequence identity with any one of SEQ ID NOs: 8 to 14; (iv) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (i) to (iii) or their complements; and (v) a complementary sequence of any of the polynucleotides (i) to (iii).

[0096] Any of the polynucleotides (ii) to (iv) maintains the function of the original SEQ ID NOs: 8 to 14, and is intended to encode the direct repeat (DR) sequence of crRNA in the Cas13e or Cas13f system of the present invention.

[0097] As used herein, "direct repeat sequence" may refer to the DNA coding sequence in a CRISPR locus or the RNA encoded thereby in a crRNA. Thus, when any of SEQ ID NOs: 8-14 are referred to in the context of an RNA molecule, such as a crRNA, it is understood that each T represents a U.

[0098] Thus, in a particular embodiment, the isolated polynucleotide is a DNA encoding the DR sequence for the crRNA of the Cas13e and Cas13f systems of the present invention.

[0099] In certain other embodiments, the isolated polynucleotide is an RNA that is a DR sequence for the crRNA of the Cas13e and Cas13f systems of the invention.

[0100] In a fifth aspect, the present invention provides a complex comprising: (i) a protein composition, which may be any one of the Cas13e or Cas13f effector proteins of the present invention, or an orthologue, homologue, derivative, conjugate, functional fragment, conjugate, or fusion thereof; and (ii) a polynucleotide composition comprising an isolated polynucleotide as described in the fourth aspect of the present invention (e.g., a DR sequence) and a spacer sequence complementary to at least a portion of a target RNA. In certain embodiments, the DR sequence is at the 3' end of the spacer sequence.

[0101] In some embodiments, the polynucleotide composition is a guideRNA / crRNA of the Cas13e or Cas13f system of the present invention, without tracrRNA.

[0102] In certain embodiments, for use with Cas13e and Cas13f effector proteins, or their homologs, orthologs, derivatives, fusions, conjugates, or functional fragments having RNase activity, the spacer sequence is at least about 10 nucleotides, or 10-60, 15-50, 20-50, 25-40, 25-50, or 19-50 nucleotides. In certain embodiments, for use with Cas13e and Cas13f effector proteins, or their homologs, orthologs, derivatives, fusions, conjugates, or functional fragments having no RNase activity but the ability to bind to a guide RNA and a target RNA complementary to the guide RNA, the spacer sequence is at least about 10 nucleotides, or about 10-200, 15-180, 20-150, 25-125, 30-110, 35-100, 40-80, 45-60, 50-55, or about 50 nucleotides.

[0103] In certain embodiments, the DR sequence is 15-36, 20-36, 22-36, or about In certain embodiments, the DR sequence in the guide RNA has substantially the same secondary structure (including stems, bulges, and loops) as the RNA form of any one of SEQ ID NOs: 8 to 14.

[0104] In certain embodiments, the guide RNA is about 36 nucleotides longer than any of the above spacer sequence lengths, for example, 45-96, 55-86, 60-86, 62-86, or 63-86 nucleotides.

[0105] In a sixth aspect, the present invention provides an isolated polynucleotide comprising: (i) a polynucleotide encoding any one of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1 to 7, or an ortholog, homologue, derivative, functional fragment, or fusion thereof; (ii) a polynucleotide of any one of SEQ ID NOs: 8 to 14; or (iii) a polynucleotide comprising (i) and (ii).

[0106] In some embodiments, the polynucleotides are not naturally occurring / non-naturally occurring, with the exception of SEQ ID NOs: 15-21, etc.

[0107] In some embodiments, the polynucleotides are codon optimized for expression in prokaryotes. In some embodiments, the polynucleotides are codon optimized for expression in eukaryotes, such as humans or human cells.

[0108] In a seventh aspect, the present invention provides a vector comprising or containing any of the polynucleotides of the sixth aspect. The vector may be a cloning vector or an expression vector. The vector may be a plasmid, a phagemid, or a cosmid, to name just a few. In certain embodiments, the vector may be used to express a polynucleotide in a mammalian cell, such as a human cell, any one of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1-7, or an ortholog, homolog, derivative, functional fragment, fusion thereof; or any of the polynucleotides of the fourth aspect; or any of the complexes of the fifth aspect.

[0109] In an eighth aspect, the present invention provides a host cell comprising any of the polynucleotides of the fourth or sixth aspects and / or the vector of the seventh aspect of the invention. The host cell may be a cell derived from a prokaryote, such as E. coli, or a eukaryote, such as yeast, insect, plant, animal (e.g. mammalian, including human and mouse). The host cell may be an isolated primary cell (such as a bone marrow cell for ex vivo therapy), or an established cell line, such as a tumour cell line, 293T cells, or stem cells, iPCs.

[0110] In a related aspect, the invention provides a eukaryotic cell comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex, the CRISPR-Cas complex comprising: (1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA, and a direct repeat (DR) sequence 3' to the spacer sequence; and (2) a CRISPR-associated protein (Cas) having an amino acid sequence of any one of SEQ ID NOs: 1-7, or a derivative or functional fragment of said Cas; wherein the Cas, derivative, and functional fragment are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.

[0111] In a ninth aspect, the present invention provides a first (protein) composition selected from any one of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1 to 7, or orthologues, homologues, derivatives, conjugates, functional fragments, fusions thereof; and (ii) RNA, including guideRNA / crRNA, in particular spacer sequences therefor, or a second (nucleotide) composition comprising a coding sequence. The guide RNA may comprise a DR sequence and a spacer sequence that is complementary to or can hybridize with the target RNA. The guide RNA may form a complex with the first (protein) composition of (i). In some embodiments, the DR sequence may be a polynucleotide of the fourth aspect of the invention. In some embodiments, the DR sequence may be at the 3' end of the guide RNA. In some embodiments, the composition (such as (i) and / or (ii)) is non-natural or modified from a natural composition. In some embodiments, at least some components of the composition are non-natural or modified from a natural component of the composition. In some embodiments, the target sequence is RNA from a prokaryote or eukaryote, such as a non-naturally occurring RNA. The target RNA may be present inside a cell, such as in the cytosol or inside an organelle. In some embodiments, the protein composition may have an NLS, which may be located at its N-terminus or C-terminus, or inside.

[0112] In a tenth aspect, the present invention provides a composition comprising one or more vectors of the seventh aspect of the invention, the one or more vectors comprising: (i) a first polynucleotide encoding any one of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1-7, or an orthologue, homologue, derivative, functional fragment, fusion thereof, optionally operably linked to a first regulatory element; and (ii) a second polynucleotide encoding a guide RNA of the invention, optionally operably linked to a second regulatory element. The first and second polynucleotides may be on different vectors or on the same vector. The guide RNA comprises a DR sequence (such as any one of the fourth aspect) and a spacer sequence capable of forming a complex with the protein product encoded by the first polynucleotide and binding / complementing the target RNA. In some embodiments, the first regulatory element is a promoter, such as an inducible promoter. In some embodiments, the second regulatory element is a promoter, such as an inducible promoter. In some embodiments, the composition (such as (i) and / or (ii)) is non-natural or modified from a natural composition. In some embodiments, at least some components of the composition are non-natural or modified from a natural component of the composition. In some embodiments, the target sequence is RNA from a prokaryote or eukaryote, such as a non-naturally occurring RNA. The target RNA may be present inside a cell, such as in the cytosol or inside an organelle. In some embodiments, the protein composition may have an NLS, which may be located at its N-terminus or C-terminus, or internally.

[0113] In some embodiments, the vector is a plasmid. In some embodiments, the vector is a retrovirus, a replication-deficient retrovirus, an adenovirus, a replication-deficient adenovirus, or an AAV-based viral vector. In some embodiments, the vector is capable of autonomous replication in a host cell (e.g., has a bacterial origin of replication sequence). In some embodiments, the vector can integrate into and replicate in the host genome. In some embodiments, the vector is a cloning vector. In some embodiments, the vector is an expression vector.

[0114] The present invention further provides a delivery composition for delivering any of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1 to 7 of the first to third aspects of the invention, or orthologues, homologues, derivatives, conjugates, functional fragments, fusions thereof; the polynucleotide of the fourth and / or sixth aspects of the invention; the complex of the fifth aspect of the invention; the vector of the seventh aspect of the invention; the cell of the eighth aspect of the invention, and the composition of the ninth and / or tenth aspects of the invention. Delivery may be by transfection, lipofection, electroporation, gene gun, microinjection, sonication, calcium phosphate transfection, cationic transfection, viral vectors, using a vehicle such as a liposome, nanoparticle, exosome, microvesicle, gene gun, or one or more viral vectors. This can be done by any one of the methods known in the art, such as vector delivery.

[0115] The present invention further provides kits comprising any one or more of the following: any of the Cas13e or Cas13f effector proteins of SEQ ID NOs: 1-7 of the first to third aspects of the invention, or orthologues, homologues, derivatives, conjugates, functional fragments, fusions thereof; a polynucleotide of the fourth and / or sixth aspects of the invention; a complex of the fifth aspect of the invention; a vector of the seventh aspect of the invention; a cell of the eighth aspect of the invention, and a composition of the ninth and / or tenth aspects of the invention. In some embodiments, the kit may further comprise instructions on how to use the components of the kit and / or how to obtain additional components from a third party for use with the components of the kit. Any components of the kit may be stored in any suitable container.

[0116] Although the present invention has been generally described hereinabove, a more detailed description of various aspects of the present invention will be provided in the following separate sections.However, for the sake of brevity and to reduce redundancy, it should be understood that a particular embodiment of the present invention will be described in only one section, or in only claims or examples.Therefore, it should also be understood that any one embodiment of the present invention, including those described in only one aspect, section, or in only claims or examples, can be combined with any other embodiment of the present invention, unless specifically denied or the combination is inappropriate.

[0117] 2. Novel class 2, type VI CRISPR RNA-guided RNases and their derivatives In one aspect, the invention described herein provides two novel families of CRISPR class 2, type VI effectors that have two strictly conserved RX4-6H (RXXXXH) motifs characteristic of the nucleotide-binding (HEPN) domains of higher eukaryotes and prokaryotes. Similar CRISPR class 2, type VI effectors containing two HEPN domains have been previously characterized, including, for example, CRISPR Cas13a (C2c2), Cas13b, Cas13c, and Cas13d.

[0118] The HEPN domain is an RNase domain, and has been shown to confer the ability to bind and cleave target RNA molecules. Target RNA can be any suitable form of RNA, including, but not limited to, mRNA, tRNA, ribosomal RNA, non-coding RNA, lncRNA (long non-coding RNA), and nuclear RNA. For example, in some embodiments, Cas proteins recognize and cleave RNA targets located in the coding strand of an open reading frame (ORF).

[0119] In one embodiment, the present disclosure provides two families of CRISPR class 2, type VI effectors, generally referred to herein as type VI-E and type VI-F CRISPR-Cas effector proteins, Cas13e or Cas13f. Direct comparison of type VI-E and type VI-F CRISPR-Cas effector proteins with effectors from these other systems shows that type VI-E and type VI-F CRISPR-Cas effector proteins are significantly smaller (e.g., about 20% fewer amino acids) than the smallest previously identified type VI-D / Cas13d effector (see FIG. 4), and share less than 30% sequence similarity in a one-to-one sequence alignment with other previously described effector proteins, including their closest phylogenetic relative, Cas13b (see FIG. 3).

[0120] These two newly identified families of CRISPR class 2, type VI effectors can be used in a variety of applications and are particularly suitable for therapeutic applications because they are much smaller than other effectors (e.g., CRISPR Cas13a, Cas13b, Cas13c, and Cas13d effectors) and therefore can be easily integrated into AAV vectors, etc. This allows packaging of the nucleic acids encoding the effectors and their guide RNA coding sequences into a delivery system with size limitations. Furthermore, the lack of detectable collateral / non-specific RNase activity in the selected range of spacer sequence lengths (such as about 30 nucleotides, see FIG. 11 ) makes these Cas effectors less susceptible to potentially dangerous (in the absence of immunity) general off-target RNA digestion in target cells that are desired not to be destroyed after activation of specific RNase activity. On the other hand, at other selected spacer lengths such as about 30 nucleotides, significant collateral RNase activity is present for these Cas effectors, and therefore the Cas effectors of the present invention can also be used in practical applications that rely on such collateral RNase activity.

[0121] In bacteria, the VI-E and VI-F type CRISPR-Cas systems contain a single effector (approximately 775 and 790 residues, respectively) in close proximity to the CRISPR array (see FIG. 1). The CRISPR array contains a direct repeat (DR) sequence, typically 36 nucleotides long, that is generally well conserved in both sequence and secondary structure (see FIG. 2).

[0122] The data presented herein demonstrate that crRNA is processed from the 5' end such that the DR sequence terminates at the 3' end of the mature crRNA.

[0123] Spacers included in Cas13e and Cas13f CRISPR arrays are most commonly 30 nucleotides long, with most of the length variation falling within the range of 29-30 nucleotides. However, a wide range of spacer lengths can be tolerated. For example, for use in functional Cas13e or Cas13f effector proteins, or homologs, orthologs, derivatives, fusions, conjugates, or functional fragments thereof, the spacer can be 10-60 nucleotides, 20-50 nucleotides, 25-45 nucleotides, 25-35 nucleotides, or about 27, 28, 29, 30, 31, 32, or 33 nucleotides. However, for use in any of the above dCas forms, the spacer can be 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.

[0124] Exemplary type VI-E and VI-F CRISPR-Cas effector proteins are shown in the table below.

[0125] [Table 1]

[0126] [Table 2]

[0127] [Table 3]

[0128] In the above sequence, the two RX4-6H (RXXXXH) motifs in each effector are double underlined. In Cas13e.1, the C-terminal motif can have two possibilities due to the RR and HH sequences flanking the motif. Mutations in one or both such domains can generate RNase-dead forms (or "dCas") of Cas13e and Cas13f effector proteins, their homologs, orthologs, fusions, conjugates, derivatives, or functional fragments, while substantially maintaining their ability to bind to guide RNAs and target RNAs complementary to the guide RNAs.

[0129] The corresponding DR coding sequences for the Cas effectors are listed below:

[0130] [Table 4]

[0131] Because the secondary structure of a DR sequence, including the location and size of steps, bulges, and loop structures, is likely more important than the particular nucleotide sequence that forms such secondary structure, alternative or derivative DR sequences may also be used in the systems and methods of the invention, so long as these derivative or alternative DR sequences have a secondary structure substantially similar to the secondary structure of an RNA encoded by any one of SEQ ID NOs: 8-14. For example, derivative DR sequences may have ±1 or 2 base pairs in one or both stems (see FIG. 2), may have ±1, 2, or 3 bases in either or both of the single strands in the bulge, and / or may have ±1, 2, 3, or 4 bases in the loop region.

[0132] In some embodiments, type VI-E and VI-F CRISPR-Cas effector proteins include "derivatives" having an amino acid sequence with at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 1-7 above. Such derivative Cas effectors sharing significant protein sequence identity to any one of SEQ ID NOs: 1-7 retained at least one of the functions of the Cas of SEQ ID NOs: 1-7 (see below), e.g., the ability to bind to and form a complex with a crRNA comprising at least one of the DR sequences of SEQ ID NOs: 8-14. For example, a Cas13e.1 derivative may share 85% amino acid sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, or 7, respectively, and retain the ability to bind to and form a complex with a crRNA having a DR sequence of SEQ ID NO:8, 9, 10, 11, 12, 13, or 14, respectively.

[0133] In some embodiments, the derivatives contain conservative amino acid residue substitutions, hi some embodiments, the derivatives contain only conservative amino acid residue substitutions (i.e., all amino acid substitutions in the derivative are conservative substitutions and no non-conserved substitutions).

[0134] In some embodiments, the derivatives include insertions or deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less amino acids into any one of the wild-type sequences of SEQ ID NOs: 1-7. The insertions and / or deletions may be grouped together or separated over the entire length of the sequence, so long as at least one of the functions of the wild-type sequence is preserved. Such functions may include the ability to bind to the guide / crRNA, RNase activity, the ability to bind to and / or cleave a target RNA complementary to the guide / crRNA. In some embodiments, the insertions and / or deletions are not in the RXXXXH motif or within 5, 10, 15, or 20 residues from the RXXXXH motif.

[0135] In some embodiments, the derivatives have the ability to bind to guideRNA / crRNA. was holding.

[0136] In some embodiments, the derivatives retained guide / crRNA-activating RNase activity.

[0137] In some embodiments, the derivatives retained the ability to bind and / or cleave the target RNA in the presence of a bound guide / crRNA that is complementary in sequence to at least a portion of the target RNA.

[0138] In other embodiments, the derivative has completely or partially lost guide / crRNA-activating RNase activity, e.g., due to mutations in one or more catalytic residues of the RNA-guided RNase, and such derivatives may be referred to as dCas, e.g., dCas13e.1.

[0139] Thus, in certain embodiments, the derivatives may be modified to have reduced nuclease / RNase activity, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation, as compared to the corresponding wild-type protein. Nuclease activity may be reduced by several methods known in the art, e.g., by introducing mutations into the nuclease (catalytic) domain of the protein. In some embodiments, catalytic residues for nuclease activity are identified, and these amino acid residues may be substituted with different amino acid residues (e.g., glycine or alanine) to reduce nuclease activity. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the amino acid substitutions are non-conservative amino acid substitutions.

[0140] In some embodiments, the modifications include one or more mutations (e.g., amino acid deletions, insertions, or substitutions) in at least one HEPN domain, hi some embodiments, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid substitutions in at least one HEPN domain. For example, in some embodiments, 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:1, or R97, H102, R770, H775 of SEQ ID NO:2, or R77, H82, R764, H769 of SEQ ID NO:3, or R79, H84, R766A, H771 of SEQ ID NO:4, or R79, H84, R766, H771 of SEQ ID NO:5, or R89, H94, R773, H778 of SEQ ID NO:6, or R89, H94, R777, H782 of SEQ ID NO:7.

[0141] In certain embodiments, the one or more mutations or two or more mutations may be in the catalytically active domain of the effector protein that comprises the HEPN domain or a catalytically active domain homologous to the HEPN domain. In certain embodiments, the effector protein comprises one or more of the following mutations: R84A, H89A, R739A, H744A, R740A, H745A (wherein the amino acid positions correspond to those of Cas13e.1). Those skilled in the art will understand that the corresponding amino acid positions in different Cas13e and Cas13f proteins can be mutated to have the same effect. In certain embodiments, the one or more mutations completely or partially abolish the catalytic activity of the protein (e.g., altered cleavage rate, altered specificity, etc.).

[0142] Other exemplary (catalytic) residue mutations include R97A, H102A, R770A, H775A in Cas13e.2, or R77A, H82A, R764A, H769A in Cas13f.1, or R79A, H84A, R766A, H771A in Cas13f.2, or or R89A, H94A, R773A, H778A in Cas13f.4, or R89A, H94A, R777A, H782A in Cas13f.5. In certain embodiments, any of the R and / or H residues herein may be substituted with G, V, or I instead of A.

[0143] The presence of at least one of these mutations results in a derivative that has reduced or decreased RNase activity compared to the corresponding wild-type protein lacking the mutation.

[0144] In certain embodiments, the effector proteins described herein are "dead" effector proteins, such as dead Cas13e or Cas13f effector proteins (i.e., dCas13e and dCas13f). In certain embodiments, the effector proteins have one or more mutations in HEPN domain 1 (N-terminus). In certain embodiments, the effector proteins have one or more mutations in HEPN domain 2 (C-terminus). In certain embodiments, the effector proteins have one or more mutations in HEPN domain 1 and HEPN domain 2.

[0145] Inactivated Cas or derivatives or functional fragments thereof may be fused or associated with one or more heterologous / functional domains (e.g., via fusion proteins, linker peptides, "GS" linkers, etc.). These functional domains may have various activities, such as methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional 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). In some embodiments, the functional domains are Krüpper-associated box (KRAB), SID (e.g., SID4X), VP64, VPR, VP16, Fok1, P65, HSF1, adenosine deaminase acting on RNA such as MyoD1, ADAR1, ADAR2, APOBEC, cytidine deaminase (AID), TAD, mini-SOG, APEX, and biotin-APEX.

[0146] In some embodiments, the functional domain is a base editing domain, such as ADAR1 (including wild-type or its ADAR1DD form, with or without E1008Q), ADAR2 (including wild-type or its ADAR2DD form, with or without the E488Q mutation), APOBEC, or AID.

[0147] In some embodiments, 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 domains may be located at or near or near the end of the effector protein (e.g., Cas13e / Cas13f effector protein), and in the case of two or more NLSs, each of the two may be located at or near or near the end of the effector protein (e.g., Cas13e / Cas13f effector protein).

[0148] In some embodiments, at least one or more heterologous functional domains may be at or near the amino terminus of the effector protein, and / or at least one or more heterologous functional domains may be at or near the carboxy terminus of the effector protein. One or more heterologous functional domains may be fused to the effector protein. One or more heterologous functional domains may be tethered to the effector protein. One or more heterologous functional domains may be linked to the effector protein by a linker moiety.

[0149] In some embodiments, there are multiple (eg, 2, 3, 4, 5, 6, 7, 8, or more) identical or different functional domains.

[0150] In some embodiments, the functional domain (e.g., a base editing domain) is further fused to an RNA binding domain (e.g., MS2).

[0151] In some embodiments, the functional domains are associated with or fused through a linker sequence (e.g., a flexible or rigid linker sequence). Exemplary linker sequences and functional domain sequences are provided in the table below.

[0152] [Table 5]

[0153] The positioning of one or more functional domains in an inactivated Cas protein allows The functional domain is capable of proper spatial orientation so as to provide a functional effect on the target that is attributable to the functional domain. For example, if the functional domain is a transcription activator (e.g., VP16, VP64, or p65), the transcription activator is positioned in a spatial orientation that allows it to affect the transcription of the target. Similarly, a transcription repressor is positioned to affect the transcription of the target, and a nuclease (e.g., Fok1) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is positioned at the N-terminus of the Cas / dCas. In some embodiments, the functional domain is positioned at the C-terminus of the Cas / dCas. In some embodiments, the inactivated CRISPR-associated protein (dCas) is modified to include a first functional domain at the N-terminus and a second functional domain at the C-terminus.

[0154] Various examples of inactivated CRISPR-associated proteins fused to one or more functional domains and methods of using same are described, for example, in WO 2017 / 219027, which is incorporated by reference in its entirety, particularly with respect to the features described herein.

[0155] In some embodiments, the type VI-E and VI-F CRISPR-Cas effector proteins comprise the amino acid sequence of any one of SEQ ID NOs: 1-7 above. In some embodiments, the type VI-E and VI-F CRISPR-Cas effector proteins exclude the native amino acid sequence of any one of SEQ ID NOs: 1-7 above.

[0156] In some embodiments, instead of using the full-length wild-type (SEQ ID NOs: 1-7) or derivative VI-E and VI-F type Cas effectors, "functional fragments" thereof may be used.

[0157] A "functional fragment" as used herein refers to a fragment of the wild-type protein of any one of SEQ ID NOs: 1-7, or a derivative thereof having a sequence less than the full length. The deleted residues in a functional fragment may be at the N-terminus, C-terminus, and / or internal. A functional fragment retains at least one function of wild-type VI-E or VI-F Cas, or at least one function of a derivative thereof. Thus, a functional fragment is specifically defined with respect to that function. For example, a functional fragment whose function is the ability to bind crRNA and target RNA may not be a functional fragment with respect to RNase function, since losing the RXXXXH motifs at both ends of Cas does not affect its ability to bind crRNA and target RNA, but may abolish and destroy RNase activity.

[0158] In some embodiments, the type VI-E or VI-F CRISPR-Cas effector protein or derivative or functional fragment thereof lacks about 30, 60, 90, 120, 150, or about 180 residues from the N-terminus compared to the full-length sequences of SEQ ID NOs: 1-7.

[0159] In some embodiments, the type VI-E or VI-F CRISPR-Cas effector protein or derivative or functional fragment thereof lacks about 30, 60, 90, 120, or about 150 residues from the C-terminus compared to the full-length sequences of SEQ ID NOs: 1-7.

[0160] In some embodiments, compared to the full-length sequences of SEQ ID NOs: 1-7, the type VI-E or VI-F CRISPR-Cas effector protein or derivative or functional fragment thereof lacks about 30, 60, 90, 120, 150, or about 180 residues from the N-terminus and about 30, 60, 90, 120, or about 150 residues from the C-terminus.

[0161] In some embodiments, the VI-E or VI-F type CRISPR-Cas effector The terprotein or a derivative or a functional fragment thereof has RNase activity, for example, guide / crRNA activation specific RNase activity.

[0162] In some embodiments, the type VI-E or VI-F CRISPR-Cas effector protein or derivative or functional fragment thereof has no substantial / detectable collateral RNase activity.

[0163] As used herein, "collateral RNase activity" refers to the non-specific RNase activity observed in certain other class 2, type VI RNA-guided RNases, such as Cas13a. Complexes containing Cas13a undergo a conformational change following activation, e.g., by binding to a target nucleic acid (e.g., a target RNA), such that the complex now acts as a non-specific RNase and cleaves and / or degrades nearby RNA molecules (e.g., ssRNA or dsRNA molecules) (i.e., the "collateral" effect).

[0164] In certain embodiments, complexes composed of (but not limited to) type VI-E or VI-F CRISPR-Cas effector proteins or derivatives or functional fragments thereof and crRNA do not exhibit collateral RNase activity after target recognition. This "collateral-free" embodiment may include wild-type, engineered / derivative effector proteins, or functional fragments thereof.

[0165] In some embodiments, a type VI-E or VI-F CRISPR-Cas effector protein or derivative or functional fragment thereof recognizes and cleaves a target RNA without the additional requirement to be adjacent or proximal to a protospacer (i.e., a protospacer adjacent motif "PAM" or protospacer adjacent sequence "PFS" requirement).

[0166] The present disclosure also provides split forms of the CRISPR-associated proteins described herein (e.g., VI-E or VI-F type CRISPR-Cas effector proteins). Split forms of CRISPR-associated proteins can be advantageous for delivery. In some embodiments, the CRISPR-associated protein is split into two portions of an enzyme that together substantially comprise a functional CRISPR-associated protein.

[0167] Splitting can be done in a way that the catalytic domain is not affected. CRISPR-associated proteins can function as nucleases or can be inactivating enzymes, which are essentially RNA-binding proteins with little or no catalytic activity (e.g., due to mutations in their catalytic domains). Splitting enzymes are described, for example, 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.

[0168] For example, in some embodiments, the nuclease lobe and the α-helical lobe are expressed as separate polypeptides. The lobes do not interact with each other by themselves, but the crRNA recruits them into a ternary complex, which recapitulates the activity of full-length CRISPR-associated proteins and catalyzes site-specific DNA cleavage. The use of modified crRNA abolishes the degradative enzyme activity by preventing dimerization, allowing the generation of an inducible dimerization system.

[0169] In some embodiments, the split CRISPR-associated protein can be fused to a dimerization partner, for example, by using a rapamycin-sensitive dimerization domain. This allows for the generation of chemically inducible CRISPR-associated proteins for temporal control of the activity of the protein. Thus, the CRISPR-associated protein can be split into two fragments. and the rapamycin-sensitive dimerization domain can be used for controlled reorganization of the protein.

[0170] Split points are typically designed in silico and cloned into the construct. During this process, mutations can be introduced into the split CRISPR-associated protein and non-functional domains can be removed.

[0171] In some embodiments, the two portions or fragments of a split CRISPR-associated protein (i.e., an N-terminal and a C-terminal fragment) may form a complete CRISPR-associated protein that contains, for example, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the sequence of a wild-type CRISPR-associated protein.

[0172] The CRISPR-associated proteins described herein (e.g., VI-E or VI-F type CRISPR-Cas effector proteins) can be designed to be self-activating or self-inactivating. For example, a target sequence can be introduced into the coding construct of the CRISPR-associated protein. Thus, the CRISPR-associated protein can cleave the target sequence as well as the coding construct of the protein, thereby self-inactivating their expression. Methods for constructing self-inactivating CRISPR systems are described, for example, in Epstein and Schaffer, Mol. Ther. 24:S50, 2016, which is incorporated herein by reference.

[0173] In some other embodiments, an additional crRNA expressed under the control of a weak promoter (e.g., 7SK promoter) may target a nucleic acid sequence encoding a CRISPR-associated protein and prevent and / or block its expression (e.g., by preventing transcription and / or translation of the nucleic acid). Transfection of a cell with a vector expressing a CRISPR-associated protein, a crRNA, and a crRNA targeting a nucleic acid encoding a CRISPR-associated protein may result in efficient destruction of the nucleic acid encoding the CRISPR-associated protein, reducing the levels of the CRISPR-associated protein and thereby limiting genome editing activity.

[0174] In some embodiments, genome editing activity of CRISPR-associated proteins can be regulated through endogenous RNA signatures (e.g., miRNAs) in mammalian cells. CRISPR-associated protein switches can be made by using miRNA complementary sequences in the 5'-UTR of mRNAs encoding CRISPR-associated proteins. The switches respond selectively and efficiently to miRNAs in target cells. Thus, the switches can differentially control genome editing by sensing endogenous miRNA activity in heterogeneous cell populations. Thus, the switch system can provide a framework for cell type-selective genome editing and cell engineering based on intracellular miRNA information (see, for example, Hirosawa et al., Nucl.Acids Res.45(13):e118,2017).

[0175] CRISPR-associated proteins (e.g., VI-E and VI-F type CRISPR-Cas effector proteins) can be inducibly expressed, e.g., their expression can be light- or chemically inducible. This mechanism allows for activation of functional domains in the CRISPR-associated proteins. Light inducibility can be achieved by a variety of methods known in the art, e.g., by designing a fusion complex in which CRY2 PHR / CIBN pairing is used to split the CRISPR-associated protein (see, e.g., Konermann et al., "Optical control of mammalian endogenous transcription and epigenetic states", Nature 500:7463, 2013).

[0176] Chemical inducibility can be achieved, for example, by designing a fusion complex in which FKBP / FRB (FK506 binding protein / FKBP rapamycin binding domain) pairing is used to split a CRISPR-associated protein. Rapamycin is required to form the fusion complex, thereby activating the CRISPR-associated protein (see, e.g., Zetsche et al., "A split-Cas9 architecture for inducible genome editing and transcription modulation", Nature Biotech. 33:2:139-42, 2015).

[0177] Additionally, expression of CRISPR-associated proteins can be regulated by inducible promoters, such as tetracycline- or doxycycline-controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone-inducible gene expression systems (e.g., ecdysone-inducible gene expression systems), and arabinose-inducible gene expression systems. When delivered as RNA, expression of RNA-targeted effector proteins can be regulated via riboswitches that can sense small molecules such as 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).

[0178] Various embodiments of inducible CRISPR-associated proteins and inducible CRISPR systems are described, for example, in U.S. Pat. No. 8,871,445, U.S. Patent Application Publication No. 2016 / 0208243, and WO 2016 / 205764, each of which is incorporated by reference in its entirety.

[0179] In some embodiments, a CRISPR-associated protein comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nuclear localization signal (NLS) attached to the N-terminus or C-terminus of the protein. Non-limiting examples of NLSs include NLS sequences derived from: the SV40 virus large T-antigen NLS having the amino acid sequence PKKKRKV; an NLS derived from the nucleoplasm (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the IBB domain from importin-α, sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; the sarcoma T protein sequences VSRKRPRP and PPKKARED; the human p53 sequence PQPKKKPL; the mouse c-abl IV sequence SALIKKKKKMAP; influenza virus NS1 sequences DRLRR and PKQKKRK; delta hepatitis virus antigen sequence RKLKKKIKKL; mouse Mx1 protein sequence REKKKFLKRR; human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; and human glucocorticoid receptor sequence RKCLQAGMNLEARKTKK. In some embodiments, 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 to the N-terminus or C-terminus of the protein. In a preferred embodiment, the C-terminus and / or N-terminus NLS or NES are attached for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.

[0180] In some embodiments, the CRISPR-associated proteins described herein are mutated at one or more amino acid residues to alter one or more functional activities.

[0181] For example, in some embodiments, a CRISPR-associated protein is mutated at one or more amino acid residues to alter its helicase activity.

[0182] In some embodiments, a CRISPR-associated protein is mutated at one or more amino acid residues to alter its nuclease activity (e.g., endonuclease activity or exonuclease activity).

[0183] In some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with a guide RNA.

[0184] In some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid.

[0185] In some embodiments, the CRISPR-associated proteins described herein are capable of cleaving a target RNA molecule.

[0186] In some embodiments, the CRISPR-associated protein is mutated in one or more amino acid residues to modify its cleavage activity. For example, in some embodiments, the CRISPR-associated protein may contain one or more mutations that cause the enzyme to be unable to cleave the target nucleic acid.

[0187] In some embodiments, the CRISPR-associated protein is capable of cleaving the strand of a target nucleic acid that is complementary to the strand to which the guide RNA hybridizes.

[0188] In some embodiments, the CRISPR-associated proteins described herein can be engineered to have deletions 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 functionally interact with a guide RNA). Truncated CRISPR-associated proteins can be advantageously used in combination with delivery systems that have load limitations.

[0189] In some embodiments, the CRISPR-associated proteins described herein may be fused to one or more peptide tags, including a His-tag, a GST-tag, a V5-tag, a FLAG-tag, an HA-tag, a VSV-G-tag, a Trx-tag, or a myc-tag.

[0190] In some embodiments, the CRISPR-associated proteins described herein may 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).

[0191] In some embodiments, the CRISPR-associated proteins described herein may be fused to MBP, a LexA DNA binding domain, or a Gal4 DNA binding domain.

[0192] In some embodiments, the CRISPR-associated proteins described herein may be linked or conjugated to a detectable label, such as a fluorescent dye, including FITC and DAPI.

[0193] In any of the embodiments herein, the CRISPR-related The linkage between the associated protein and the other moiety can be at the N-terminus or C-terminus of the CRISPR-associated protein, or can be internal via a covalent chemical bond. The linkage can be by any chemical bond known in the art, such as a peptide bond, a bond via the side chain of an amino acid, such as D, E, S, T, or an amino acid derivative (Ahx, β-Ala, GABA, or Ava), or a PEG bond.

[0194] 3. Polynucleotides The present invention also provides nucleic acids encoding the proteins (e.g., CRISPR-associated or accessory proteins) and guide RNAs (e.g., crRNAs) described herein.

[0195] In some embodiments, 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 a Cas, a derivative or a functional fragment thereof). In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methylcytidine, substituted with pseudouridine, or a combination thereof.

[0196] In some embodiments, the nucleic acid (e.g., DNA) is operably linked to a regulatory element (e.g., a promoter) to control expression of the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter.

[0197] Suitable promoters are known in the art, and include, for example, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, and β-actin promoter. For example, the U6 promoter can be used to regulate the expression of the guide RNA molecules described herein.

[0198] In some embodiments, the nucleic acid is present in a vector (e.g., a viral vector or a phage). The vector may be a cloning vector or an expression vector. The vector may be a plasmid, phagemid, cosmid, etc. The vector may include one or more regulatory elements that allow the vector to propagate in a cell of interest (e.g., a bacterial cell or a mammalian cell). In some embodiments, the vector includes a nucleic acid encoding a single component of the CRISPR-associated (Cas) system described herein. In some embodiments, the vector includes multiple nucleic acids, each encoding a component of the CRISPR-associated (Cas) system described herein.

[0199] In one aspect, the present disclosure provides a nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence described herein, i.e., a nucleic acid sequence encoding a Cas protein, derivative, functional fragment, or guide / crRNA comprising a DR sequence of SEQ ID NOs: 8-14.

[0200] In another aspect, 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 described herein, e.g., SEQ ID NOs:1-7.

[0201] In some embodiments, the nucleic acid sequence has 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 sequence has 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 differs from the sequences described herein.

[0202] In related embodiments, the invention provides amino acid sequences that 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 the same as the sequences described herein. In some embodiments, 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 differ from the sequences described herein.

[0203] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acids, or one or both of the first and second nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In general, the length of the 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. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences and the number of identical positions shared by the sequences, taking into account the length of each gap. For purposes of this disclosure, 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 extension penalty of 4, and a frameshift gap penalty of 5.

[0204] The proteins described herein (e.g., CRISPR-associated proteins or accessory proteins) can be delivered and used as nucleic acid molecules or polypeptides.

[0205] In certain embodiments, the nucleic acid molecule encoding the CRISPR-associated protein, its derivative or functional fragment is codon-optimized for expression in a host cell or organism. The host cell may include established cell lines (e.g., 293T cells) or isolated primary cells. The nucleic acid may be codon-optimized for use in any organism of interest, particularly human cells or bacteria. For example, the nucleic acid may be codon-optimized for any prokaryotic organism (such as E. coli), or any eukaryotic organism, such as humans, and other non-human eukaryotic organisms, including yeast, worms, insects, plants, and algae (including food crops, rice, corn, vegetables, fruits, trees, and grasses), vertebrates, fish, non-human mammals (e.g., mice, rats, rabbits, dogs, birds (such as chickens), livestock (cows, pigs, horses, sheep, goats, etc.), or non-human primates). Codon usage tables are readily available, for example at the "Codon Usage Database" available at www.kazusa.orjp / codon / , and the tables can be constructed in a number of ways. See, Akamura et al., Nucl. Acids Res. 28:292, 2000, which is incorporated herein by reference in its entirety. Computer algorithms are also available that codon-optimize a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0206] An example of a codon-optimized sequence, in this case as discussed herein, is a sequence optimized for expression in a eukaryote, such as a human (i.e., optimized for expression in a human), or for another eukaryote, animal, or mammal; see, for example, the SaCas9 human codon-optimized sequence in WO 2014 / 093622 (PCT / US2013 / 074667). While this is preferred, other examples are of course possible, and codon optimization for host species other than humans, or for specific organs, is known. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the original sequence with a codon that is more frequently or most frequently used in the genes of the host cell of interest, while maintaining the original amino acid sequence, for enhanced expression in the host cell of interest. Different species show particular biases for particular codons of particular amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA). This in turn is believed to depend, inter alia, on the properties of the codons to be translated and on the availability of particular transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is usually a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at http: / / www.kazusa.orjp / codon / , and can be constructed in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).Also, computer algorithms are available that codon-optimize a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a Cas-encoding sequence correspond to the most frequently used codon for a particular amino acid.

[0207] 4. RNA guide or crRNA In some embodiments, the CRISPR systems described herein include at least an RNA guide (e.g., a gRNA or a crRNA).

[0208] Multiple RNA guide architectures are known in the art (see, e.g., WO 2014 / 093622 and WO 2015 / 070083, the entire contents of each of which are incorporated herein by reference).

[0209] In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more RNA guides).

[0210] In some embodiments, the RNA guide comprises a crRNA. , the RNA guide comprises crRNA rather than tracrRNA.

[0211] The sequences of guide RNAs from multiple CRISPR systems are generally known in the art. See, for example, Grissa et al. (Nucleic Acids Res. 35 (web server publication): W52-7, 2007; Grissa et al., BMC Bioinformatics 8: 172, 2007; Grissa et al., Nucleic Acids Res. 36 (web server publication): W145-8, 2008; and Moller and Liang, PeerJ 5: e3788, 2017; CRISPR database at crispr.i2bc.paris-saclayfr / crispr / BLAST / CRISPRsBlast.php; and MetaCRAST available at github.com / molleraj / MetaCRAST). All are incorporated herein by reference.

[0212] In some embodiments, the crRNA comprises a direct repeat (DR) sequence and a spacer sequence. In certain embodiments, the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence, preferably at the 3' end of the spacer sequence.

[0213] Generally, the Cas protein forms a complex with the mature crRNA and the spacer sequence directs the complex to sequence-specific binding to a target RNA that is complementary to and / or hybridizes to the spacer sequence. The resulting complex comprises the Cas protein and the mature crRNA bound to the target RNA.

[0214] The direct repeat sequences for the Cas13e and Cas13f systems are generally well conserved, especially at the termini, with GCTG for Cas13e and GCTGT for Cas13f at the 5' end, and reverse complements CAGC for Cas13e and ACAGC for Cas13f at the 3' end. This conservation suggests strong base pairing for the RNA stem-loop structure that potentially interacts with proteins within the locus.

[0215] In some embodiments, the direct repeat sequence comprises the general secondary structure of 5'-S1a-Ba-S2a-L-S2b-Bb-S1b-3' when in RNA, where segments S1a and S1b are reverse complementary sequences and form a first stem (S1) having four nucleotides in Cas13e and five nucleotides in Cas13f; segments Ba and Bb are not base paired with each other and form a symmetric or nearly symmetric bulge (B) and Cas13f is not base paired with each other. Segments S2a and S2b are reverse complementary sequences and form a second stem (S2) with 5 base pairs in Cas13e and 6 or 5 base pairs in Cas13f; L is an 8-nucleotide loop in Cas13e and a 5-nucleotide loop in Cas13f. See FIG. 2.

[0216] In certain embodiments, S1a has a series of GCUG in Cas13f and a series of GCUGU in Cas13e.

[0217] In certain embodiments, S2a has a series of GCCCC in Cas13f and a series of A / G CCUC G / A in Cas13e (wherein the first A or G can be absent).

[0218] In some embodiments, the direct repeat sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:8-14.

[0219] As used herein, "direct repeat sequence" may refer to a DNA coding sequence in a CRISPR locus or to an RNA encoded by the same in a crRNA. Thus, when any of SEQ ID NOs: 8-14 are referred to in the context of an RNA molecule, such as a crRNA, it is understood that each T represents a U.

[0220] In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence having a deletion, insertion, or substitution of up to 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides of SEQ ID NOs: 8-14. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity to SEQ ID NOs: 8-14 (e.g., due to a deletion, insertion, or substitution of a nucleotide in SEQ ID NOs: 8-14). In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence that is not identical to any one of SEQ ID NOs: 8-14 but is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 8-14 or is capable of binding to the complement of any one of SEQ ID NOs: 8-14 under physiological conditions.

[0221] In certain embodiments, the deletions, insertions, or substitutions do not change the overall secondary structure of SEQ ID NOs: 8-14 (e.g., the relative positions and / or sizes of the stems, bulges, and loops do not deviate significantly from the original stems, bulges, and loops). For example, deletions, insertions, or substitutions can be made within the bulge or loop regions such that the overall symmetry of the bulge remains largely the same. Deletions, insertions, or substitutions can be made within the stems such that the total length of the stem does not deviate significantly from the original stem (e.g., adding or deleting one base pair in each of the two stems corresponds to a total of four base changes).

[0222] In certain embodiments, the deletions, insertions, or substitutions result in derivative DR sequences that may have ±1 or 2 base pairs in one or both stems (see Figure 2), may have ±1, 2, or 3 bases in one or both stems of the single strand within the bulge, and / or may have ±1, 2, 3, or 4 bases in the loop region.

[0223] In a specific embodiment, any of the above direct repeat sequences different from any one of SEQ ID NOs: 8 to 14 retains the ability to function as a DR sequence of SEQ ID NOs: 8 to 14 and as a direct repeat sequence in the Cas13e protein or the Cas13f protein.

[0224] In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having the nucleic acid sequence of any one of SEQ ID NOs: 8-14, with a truncation of the first 3, 4, 5, 6, 7, or 8 3' nucleotides.

[0225] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:1 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:8.

[0226] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:2 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:9.

[0227] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:3 and the crRNA comprises a direct repeat sequence, the direct repeat sequence being SEQ ID NO: The nucleic acid sequence of claim 1, further comprising or consisting of the nucleic acid sequence of claim 10.

[0228] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:4 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:11.

[0229] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:5 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:12.

[0230] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:6 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:13.

[0231] In some embodiments, the Cas protein comprises the amino acid sequence of SEQ ID NO:7 and the crRNA comprises a direct repeat sequence, which comprises or consists of the nucleic acid sequence of SEQ ID NO:14.

[0232] In classical CRISPR systems, the degree of complementarity between a guide sequence (e.g., crRNA) and 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 between 90-100%.

[0233] 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. For example, a spacer used for a functional Cas13e or Cas13f effector protein, or a homolog, ortholog, derivative, fusion, conjugate, or functional fragment thereof can be 10-60 nucleotides, 20-50 nucleotides, 25-45 nucleotides, 25-35 nucleotides, or about 27, 28, 29, 30, 31, 32, or 33 nucleotides. However, the spacer used in any of the above dCas versions can be 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.

[0234] To reduce off-target interactions, e.g., to reduce guide interactions with target sequences with low complementarity, mutations can be introduced into the CRISPR system so that the CRISPR system can distinguish between targets and off-target sequences with more than 80%, 85%, 90%, or 95% complementarity. In some embodiments, the degree of complementarity is 80%-95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (e.g., to distinguish a target with 18 nucleotides from an off-target of 18 nucleotides with 1, 2, or 3 mismatches). Thus, in some embodiments, 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%.

[0235] In the art, perfect complementarity is necessary if there is sufficient complementarity to be functional. It is known that the cleavage efficiency is not necessarily the same as that of the target sequence. The cleavage efficiency can be controlled by introducing one or more mismatches, such as one or two mismatches, between the spacer sequence and the target sequence (including the position of the mismatch along the spacer / target). The more centrally (i.e., not at the 3' or 5' end) the mismatch, such as a double mismatch, is located; the more the cleavage efficiency is affected. Thus, by selecting the mismatch position along the spacer sequence, the cleavage efficiency can be controlled. For example, if less than 100% cleavage of the target is desired (e.g., in a cell population), one or two mismatches between the spacer and the target sequence can be introduced into the spacer sequence.

[0236] It has been demonstrated that the type VI CRISPR-Cas effector uses multiple RNA guides, allowing the effector and systems and complexes comprising it to target multiple nucleic acids. In some embodiments, the CRISPR system described herein comprises multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or more) RNA guides. In some embodiments, the CRISPR system described herein comprises a single RNA strand, or a nucleic acid encoding a single RNA strand, where the RNA guides are arranged in tandem. The single RNA strand may comprise multiple copies of the same RNA guide, multiple copies of different RNA guides, or a combination thereof. The processing capabilities of the type VI-E and type VI-F CRISPR-Cas effector proteins described herein allow these effectors to target multiple target nucleic acids (e.g., target RNAs) without loss of activity. In some embodiments, VI-E and VI-F CRISPR-Cas effector proteins can be delivered complexed with multiple RNA guides directed to different target RNAs. In some embodiments, VI-E and VI-F CRISPR-Cas effector proteins can be delivered with multiple RNA guides, each specific to a different target nucleic acid. Methods of multiplexing with CRISPR-associated proteins are described, for example, in U.S. Pat. No. 9,790,490 B2 and European Patent No. 3009511 B1, the entire contents of each of which are expressly incorporated herein by reference.

[0237] The spacer length of the crRNA can range from about 10 to 60 nucleotides, e.g., 15 to 50 nucleotides, 20 to 50 nucleotides, 25 to 50 nucleotides, or 19 to 50 nucleotides. In some embodiments, the spacer length of the 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. In some embodiments, the spacer length is 15-17 nucleotides (e.g., 15, 16, or 17 nucleotides), 17-20 nucleotides (e.g., 17, 18, 19, or 20 nucleotides), 20-24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), 23-25 ​​nucleotides (e.g., 23, 24, or 25 nucleotides), 24-27 nucleotides, 27-30 nucleotides, 30-45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides), 30 or 35-40 nucleotides, 41-45 nucleotides, 45-50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides), or longer. In some embodiments, the spacer length is from about 15 to about 42 nucleotides.

[0238] In some embodiments, the direct repeat length of the guide RNA is 15-36 nucleotides, at least 16 nucleotides, 16-20 nucleotides (e.g., 16, 17, 18, 19, or 20 nucleotides), 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 30-40 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109, 109, 108, 109, 1 In some embodiments, the length of the direct repeat of the guide RNA is 36 nucleotides.

[0239] In some embodiments, the total length of the crRNA / guideRNA is about 36 nucleotides longer than any one of the spacer sequence lengths described herein above. For example, the total length of the crRNA / guideRNA can be 45-86 nucleotides, 60-86 nucleotides, 62-86 nucleotides, or 63-86 nucleotides.

[0240] The crRNA sequence may be modified to allow for successful formation of a complex between the crRNA and a CRISPR-associated protein and successful binding to the target while at the same time not allowing successful nuclease activity (i.e., no nuclease activity / no 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 respect to nuclease activity. Dead guide sequences are typically shorter than each guide sequence that results in active RNA cleavage. In some embodiments, the dead guide is 5%, 10%, 20%, 30%, 40%, or 50% shorter than each guide RNA with nuclease activity. The dead guide sequence of the guide RNA may be 13-15 nucleotides long (e.g., 13, 14, or 15 nucleotides long), 15-19 nucleotides long, or 17-18 nucleotides long (e.g., 17 nucleotides long).

[0241] Thus, in one aspect, the present disclosure provides a non-naturally occurring or engineered CRISPR system comprising functional CRISPR-associated proteins and crRNA as described herein, wherein the crRNA comprises a dead crRNA sequence such that the crRNA can hybridize to a target sequence without detectable nuclease activity (e.g., RNase activity) such that the CRISPR system is directed to a genomic locus of interest in a cell.

[0242] A detailed description of dead guides is provided, for example, in WO 2016 / 094872, which is incorporated herein by reference in its entirety.

[0243] Guide RNAs (e.g., crRNAs) can be generated as components of an inducible system. The inducible nature of the system allows for spatio-temporal control of gene editing or gene expression. In some embodiments, examples of stimuli for the inducible system include electromagnetic radiation, acoustic energy, chemical energy, and / or thermal energy.

[0244] In some embodiments, transcription of the guide RNA (e.g., crRNA) can be regulated by an inducible promoter, such as tetracycline or doxycycline controlled transcription 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. Other examples of inducible systems include small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), light-inducible systems (phytochrome, LOV domain, or cryptochrome), or light-inducible transcription effectors (LITEs). These inducible systems are described, for example, in WO2016205764 and US8795965, both of which are incorporated herein by reference in their entireties.

[0245] Chemical modifications can be made to the phosphate backbone, sugars, and / or bases of the crRNA. Backbone modifications such as phosphorothioate modify the charge on the phosphate backbone and aid in oligonucleotide delivery and nuclease resistance (see, e.g., Eckstein, "Phosphorothioates, essential components of therapeutic oligonucleotides", Nucl. Acid Ther., 24, pp. 374-387, 2014); sugar modifications such as 2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acid (LNA) enhance both base pairing and nuclease resistance (see, e.g., Allerson et al. "Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA", J. Med. Chem. 48.4:901-904, 2005). Chemically modified bases such as 2-thiouridine or N6-methyladenosine, among others, may allow stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering", Front. Genet., August 20, 2012;3:154). In addition, RNA is amenable to 5' and 3' end conjugation with various functional moieties, including fluorescent dyes, polyethylene glycol, or proteins.

[0246] A wide variety of modifications can be made to chemically synthesized crRNA molecules. For example, modification of oligonucleotides with 2'-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing. Furthermore, 2'-OMe modifications can affect how the oligonucleotide interacts with transfection reagents, proteins, or any other molecules in cells. The effect of such modifications can be determined by experimental testing.

[0247] In some embodiments, the crRNA contains one or more phosphorothioate modifications. In some embodiments, the crRNA contains one or more locked nucleic acids to enhance base pairing and / or increase nuclease resistance.

[0248] For a summary of these chemical modifications, see, for example, Kelley et al., “Versatility of chemically synthesized guideline RNAs for CRISPR-Cas9 genome editing”, J. Biotechnol. 233:74-83, 2016; WO 2016205764; and U.S. Pat. No. 8,795,965 B2; each of which is incorporated by reference in its entirety.

[0249] The sequence and overall length of the RNA guide (e.g., crRNA) described herein can be optimized. In some embodiments, the optimized overall length of the RNA guide can be determined by identifying the processed form of the crRNA (i.e., mature crRNA) or by experimental full-length studies of the crRNA tetraloop.

[0250] The crRNA may also contain one or more aptamer sequences. Aptamers are oligonucleotide or peptide molecules that have a specific three-dimensional structure and can bind to a specific target molecule. Aptamers may be specific for gene effectors, gene activators, or gene repressors. In some embodiments, aptamers may be specific for proteins that are specific for and recruit and / or bind to a specific gene effector, gene activator, or gene repressor. The effector, activator, or repressor may be in the form of a fusion protein. In some embodiments, aptamers may be specific for proteins that are specific for and recruit and / or bind to a specific gene effector, gene activator, or gene repressor. In some embodiments, the guide RNA has two or more aptamer sequences that are specific to the same adaptor protein. In some embodiments, the two or more aptamer sequences are specific to different adaptor proteins. Examples of adaptor proteins may include MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φkCb5, φkCb8r, φkCb12r, φkCb23r, 7s, and PRR1. Thus, in some embodiments, the aptamer is selected from a binding protein that specifically binds to any one of the adaptor proteins described herein. In some embodiments, the aptamer sequence is the MS2 binding loop (5'-ggcccAACAUGAGGAUCACCCAUGUCUGCAGgggcc-3'). In some embodiments, the aptamer sequence is the QBeta binding loop (5'-ggcccAUGCUGUCUAAGACAGCAUgggcc-3'). In some embodiments, the aptamer sequence is the PP7 binding loop (5'-ggcccUAAGGGUUUAUAUGGAAACCCUUAgggcc-3'). A detailed description of aptamers can be found, for example, in Nowak et al., "Guide RNA "Engineering for versatile Cas9 functionality", Nucl. Acid. Res., 44(20):9555-9564, 2016; and WO2016205764, which are incorporated by reference in their entireties.

[0251] In certain embodiments, the method uses chemically modified guide RNA. Examples of guide RNA chemical modifications include, but are not limited to, the incorporation of 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS), or 2'-O-methyl 3'-thio PACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs may include increased stability and increased activity compared to unmodified guide RNAs, but the on-target vs. off-target specificity is not predictable. See Hendel, Nat Biotechnol. 33(9):985-9, 2015 (incorporated by reference). Additionally, chemically modified guide RNAs may include, but are not limited to, RNAs with phosphorothioate bonds and locked nucleic acid (LNA) nucleotides, including methylene bridges between the 2' and 4' carbons of the ribose ring.

[0252] The invention also encompasses a method of delivering multiple nucleic acid components, each nucleic acid component being specific for a different target locus of interest, thereby modifying multiple target loci of interest. The nucleic acid components of the complex may include one or more protein-binding RNA aptamers. The one or more aptamers may bind to a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group including Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In a particular embodiment, the bacteriophage coat protein is MS2.

[0253] 5.Target RNA The target RNA can be any RNA molecule of interest, including naturally occurring and engineered RNA molecules. The target RNA can be an mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, microzyme, or viral RNA.

[0254] In some embodiments, the target nucleic acid is associated with a condition or disease (e.g., an infectious disease or cancer). do.

[0255] Thus, in some embodiments, the systems described herein can be used to treat conditions or diseases by targeting these nucleic acids. For example, target nucleic acids associated with a condition or disease can be RNA molecules that are overexpressed in diseased cells (e.g., cancer or tumor cells). Target nucleic acids can also be toxic RNA and / or mutant RNA (e.g., mRNA molecules with splicing defects or mutations). Target nucleic acids can also be RNA specific to a particular microorganism (e.g., pathogenic bacteria).

[0256] 6. Complexes and Cells One aspect of the invention provides a CRISPR / Cas13e or CRISPR / Cas13f complex comprising (1) any Cas13e / Cas13f effector protein described herein, or a homolog, ortholog, fusion, derivative, conjugate, or functional fragment thereof, and (2) any guide RNA described herein (each comprising a spacer sequence designed to be at least partially complementary to a target RNA), and a DR sequence compatible with the Cas13e / Cas13f effector protein, or a homolog, ortholog, fusion, derivative, conjugate, or functional fragment thereof.

[0257] In certain embodiments, the complex further comprises a target RNA that is bound by the guide RNA.

[0258] In certain embodiments, the complex is not naturally occurring / occurring. For example, at least one of the components of the complex is not naturally occurring / occurring. In certain embodiments, the Cas13e / Cas13f effector protein, its homologs, orthologs, fusions, derivatives, conjugates, or functional fragments are not naturally occurring / occurring, for example, due to the presence of at least one amino acid mutation (deletion, insertion, and / or substitution) compared to the wild-type protein. In certain embodiments, the DR sequence is not naturally occurring / occurring, i.e., not one of SEQ ID NOs: 8-14, for example, due to the addition, deletion, and / or substitution of at least one nucleotide base in the wild-type sequence. In certain embodiments, the spacer sequence is not naturally occurring in that it is not present or is not encoded by any spacer sequence present in the wild-type CRISPR locus of the prokaryote in which the subject Cas13e or Cas13f is present. A spacer sequence may not be naturally occurring if it is not 100% complementary to a naturally occurring bacteriophage nucleic acid.

[0259] In a related embodiment, the invention also provides a cell comprising any of the complexes of the invention.

[0260] In certain embodiments, the cell is a prokaryote.

[0261] In certain embodiments, the cell is a eukaryotic organism. When the cell is a eukaryotic organism, the complex in the eukaryotic cell can be the naturally occurring Cas13e / Cas13f complex in the prokaryotic organism from which Cas13e / Cas13f is isolated.

[0262] 7. Method using CRISPR system The CRISPR system described herein has a wide variety of utilities, including modifying (e.g., deleting, inserting, translocating, inactivating, or activating) target polynucleotides or nucleic acids in a multiplex of cell types. CRISPR systems are useful for, for example, DNA / RNA detection (e.g., Specific High Sensitivity Enzyme Reporter Unlock (SHERLOCK)), tracking and labeling of nucleic acids, enrichment assays (subtracting desired sequences from background), control of interfering RNA or miRNA, detection of circulating tumor DNA, preparation of next generation libraries, drug discovery, and the like. It has a wide range of applications in screening, disease diagnosis and prediction, and treatment of various genetic disorders.

[0263] DNA / RNA detection In one embodiment, the CRISPR system described herein can be used for DNA or RNA detection. As shown in the examples, the Cas13e and Cas13f proteins of the present invention exhibit non-specific / collateral RNase activity by activating their guide RNA-dependent specific RNase activity when the spacer sequence is about 30 nucleotides. Thus, the CRISPR-associated proteins of the present invention can be reprogrammed by CRISPR RNAs (crRNAs) to provide a platform for specific RNA detection. By selecting a specific spacer sequence length and by recognizing its RNA target, the activated CRISPR-associated protein engages in "collateral" cleavage of nearby non-target RNA. This crRNA-programmed collateral cleavage activity allows the CRISPR system to detect the presence of specific RNA by triggering programmed cell death or by non-specific degradation of the labeled RNA.

[0264] The SHERLOCK method (Specific High Sensitivity Enzymatic Reporter Unlocking) provides an in vitro nucleic acid detection platform with attomole sensitivity based on nucleic acid amplification and collateral cleavage of reporter RNA, allowing real-time detection of target. Detection can be combined with various isothermal amplification steps to achieve signal detection. For example, recombinase polymerase amplification (RPA) can be coupled with T7 transcription to convert amplified DNA into RNA for subsequent detection. The combination of RPA amplification, T7 RNA polymerase transcription of amplified DNA into RNA, and detection of target RNA by collateral RNA cleavage-mediated release of reporter signal is called SHERLOCK. The method of using CRISPR in SHERLOCK is described in detail, for example, in Gootenberg, et al. "Nucleic acid detection with CRISPR-Cas13a / C2c2", Science, April 28, 2017; 356(6336):438-442, which is incorporated herein by reference in its entirety.

[0265] CRISPR-associated proteins can be used in Northern blot assays, which use electrophoresis to separate RNA samples by size. CRISPR-associated proteins can be used to specifically bind and detect target RNA sequences. CRISPR-associated proteins can also be fused to fluorescent proteins (e.g., GFP) and used to track RNA localization in live cells. More specifically, CRISPR-associated proteins can be inactivated in that they no longer cleave RNA as described above. Thus, CRISPR-associated proteins can be used to determine the localization of RNA or specific splice variants, mRNA transcript levels, transcript upregulation or downregulation, and disease-specific diagnosis. CRISPR-associated proteins can be used to visualize RNA in (live) cells, for example, using fluorescence microscopy or flow cytometry, such as fluorescence-activated cell sorting (FACS), which allows high-throughput screening of cells and recovery of live cells after cell sorting. A detailed description of how to detect DNA and RNA can be found, for example, in WO 2017 / 070605, which is incorporated herein by reference in its entirety.

[0266] In some embodiments, the CRISPR systems described herein are The method can be used for Rex error-robust fluorescence in situ hybridization (MERFISH), a method described, for example, in Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells", Science, April 24, 2015;348(6233):aaa6090, which is incorporated herein by reference in its entirety.

[0267] In some embodiments, the CRISPR system described herein can be used to detect target RNA in a sample (e.g., a clinical sample, a cell, or a cell lysate). The collateral RNase activity of the VI-E and / or VI-F CRISPR-Cas effector proteins described herein is activated when the effector protein binds to a target nucleic acid when the spacer sequence is of a specific selected length (e.g., about 30 nucleotides). By binding to the target RNA of interest, the effector protein cleaves the labeled detector RNA to generate a signal (e.g., an increased or decreased signal), thereby allowing the qualitative and quantitative detection of the target RNA in the sample. Specific detection and quantification of RNA in a sample allows for a number of applications, including diagnostic methods. In some embodiments, the method comprises contacting a sample with: i) an RNA guide (e.g., crRNA), and / or a nucleic acid encoding the RNA guide; the RNA guide consists of a direct repeat sequence and a spacer sequence capable of hybridizing to a target RNA; (ii) a type VI-E or type VI-F CRISPR-Cas effector protein (Ca13e or Cas13f), and / or a nucleic acid encoding an effector protein; and (iii) a labeled detector RNA; the effector protein binds to the RNA guide to form a complex; the RNA guide hybridizes to the target RNA; upon binding of the complex to the target RNA, the effector protein exhibits collateral RNase activity to cleave the labeled detector RNA; and b) measuring a detection signal generated by cleavage of the labeled detector RNA, said measuring providing detection of single-stranded target RNA in the sample. In some embodiments, the method further comprises comparing the detection signal to a reference signal and determining the amount of target RNA in the sample. In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based detection.In some embodiments, the labeled detector RNA comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluor pair. In some embodiments, cleavage of the labeled detector RNA by the effector protein reduces or increases the amount of detection signal generated by the labeled detector RNA. In some embodiments, the labeled detector RNA generates a first detection signal before cleavage by the effector protein and a second detection signal after cleavage by the effector protein. In some embodiments, a detection signal is generated when the labeled detector RNA is cleaved by the effector protein. In some embodiments, the labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, a modified nucleobase linkage, or a combination thereof. In some embodiments, the method includes multi-channel detection of multiple independent target RNAs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or more target RNAs) in a sample using multiple types of VIE and / or VI-F CRISPR-Cas (Cas13e and / or Cas13f) systems, each containing a different orthologous effector protein and corresponding RNA guide, allowing differentiation of multiple target RNAs in a sample. In some embodiments, the method includes multi-channel detection of multiple independent target RNAs in a sample using multiple instances of VI-E and / or VI-F CRISPR-Cas systems, each containing orthologous effector proteins with distinguishable collateral RNase substrates. The method of detecting RNA in a sample using CRISPR-associated proteins includes, for example: See, for example, U.S. Patent Application Publication No. 2017 / 0362644, the entire contents of which are incorporated herein by reference.

[0268] Nucleic Acid Tracking and Labeling Cellular processes depend on a network of molecular interactions between proteins, RNA, and DNA. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques use affinity tags in combination with reporter groups, such as photoactivatable groups, to in vitro label polypeptides and RNAs in the vicinity of a protein or RNA of interest. After UV irradiation, the photoactivatable groups label the tagged molecules by reacting with proteins and other molecules in close proximity to the tagged molecules. The labeled interacting molecules can then be retrieved and identified. CRISPR-associated proteins can be used, for example, to direct probes to selected RNA sequences. The application can also be applied to animal models for in vivo imaging of diseases or difficult-to-culture cell types. Methods for tracking and labeling nucleic acids are described, for example, in U.S. Pat. No. 8,795,965, WO2016205764, and WO2017070605, each of which is incorporated herein by reference in its entirety.

[0269] RNA isolation, purification, enrichment, and / or depletion The CRISPR system (e.g., CRISPR-associated protein) described herein can be used to isolate and / or purify RNA. The CRISPR-associated protein can be fused to an affinity tag that can be used to isolate and / or purify the RNA-CRISPR-associated protein complex. This application is useful, for example, for analyzing gene expression profiles in cells.

[0270] In some embodiments, CRISPR-associated proteins can be used to target specific non-coding RNA (ncRNA) and block its activity.In some embodiments, CRISPR-associated proteins can be used to specifically enrich (including but not limited to increase stability, etc.) specific RNA, or alternatively, to specifically deplete specific RNA (e.g., specific splice variants, isoforms, etc.).

[0271] These methods are described, for example, in U.S. Pat. No. 8,795,965, WO2016205764, and WO2017070605; each of which is incorporated by reference in its entirety.

[0272] High-throughput screening The CRISPR system described herein can be used to prepare next-generation sequencing (NGS) library.For example, to generate cost-effective NGS library, CRISPR system can be used to disrupt the coding sequence of target gene, and the clones transfected with CRISPR-associated proteins can be simultaneously screened by next-generation sequencing (e.g., by Ion Torrent PGM system).Detailed description of how to prepare NGS library can be found, for example, 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.

[0273] Engineered microbes Microorganisms (e.g., E. coli, yeast, and microalgae) are widely used in synthetic biology. The development of synthetic biology has broad utility, including various clinical applications. For example, the programmable CRISPR system can be used to split proteins of toxic domains for target cell death, for example, using cancer-associated RNA as a target transcript. Furthermore, pathways involved in protein-protein interactions can be influenced in synthetic biological systems by fusion complexes with appropriate effectors, such as kinases or enzymes.

[0274] In some embodiments, crRNA targeting a phage sequence can be introduced into a microorganism. Thus, the present disclosure also provides a method for inoculating a microorganism (e.g., a production strain) for phage infection.

[0275] In some embodiments, the CRISPR system provided herein can be used to engineer microorganisms, for example, to improve yield or improve fermentation efficiency.For example, the CRISPR system described herein can be used to engineer microorganisms, for example yeast, to produce biofuel or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose from agricultural waste as a source of fermentable sugars.More specifically, the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and / or modify endogenous genes that can interfere with biofuel synthesis. These methods of engineering microorganisms are described, for example, in Verwaal et al., "CRISPR / Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae", Yeast doi:10.1002 / yea.3278, 2017; and Hlavova et al., "Improving microalgae for biotechnology-from genetics to synthetic biology", Biotechnol. Adv., 33:1194-203, 2015, both of which are incorporated by reference in their entireties.

[0276] In some embodiments, the CRISPR system provided herein can be used to induce cell (e.g., microorganism, e.g., engineered microorganism) death or quiescence. The method can be used to induce quiescence or death of many cell types, including, but not limited to, mammalian cells (e.g., cancer cells or tissue culture cells), protozoa, fungal cells, virus-infected cells, intracellular bacteria-infected cells, intracellular protozoa-infected cells, prion-infected cells, prokaryotic and eukaryotic cells, including bacteria (e.g., pathogenic and non-pathogenic bacteria), protozoa, and unicellular and multicellular parasites. For example, in the field of synthetic biology, it is highly desirable to have a mechanism to control engineered microorganisms (e.g., bacteria) to prevent growth or spread. The system described herein can be used as a "kill-switch" to regulate and / or prevent the growth or spread of engineered microorganisms. Additionally, there is a need in the art for alternatives to current antibiotic treatments. The system described herein can also be used in applications where it is desired to kill or control a particular microbial population (e.g., bacterial population). For example, the systems described herein may include an RNA guide (e.g., crRNA) that targets a genus-, species-, or strain-specific nucleic acid (e.g., RNA) and can be delivered to a cell. Upon complexing and binding to the target nucleic acid, the collateral RNase activity of the type VI-E and / or type VI-F CRISPR-Cas effector proteins is activated, leading to cleavage of non-target RNA within the microorganism, ultimately resulting in dormancy or death. In some embodiments, the methods include injecting cells with type VI-E and / or type VI-F CRISPR-Cas effector proteins, or nucleic acids encoding the effector proteins, and RNase activity. A guide (e.g., crRNA), or a system described herein comprising a nucleic acid encoding an 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 the target nucleic acid (e.g., genus, strain, or species specific RNA guide). Without wishing to be bound by any particular theory, cleavage of non-target RNA by Type VI-E and / or Type VI-F CRISPR-Cas effector proteins may induce programmed cell death, cytotoxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, reduced cell growth, or reduced cell proliferation. For example, in bacteria, cleavage of non-target RNA by type VI-E and / or type VI-F CRISPR-Cas effector proteins can be bacteriostatic or bacteriocidal.

[0277] Uses in plants The CRISPR system described herein has a wide variety of uses in plants.In some embodiments, the CRISPR system can be used to manipulate the genome of a plant (e.g., to improve production, to produce products with desired post-translational modifications, or to introduce genes to produce industrial products).In some embodiments, the CRISPR system can be used to introduce desired traits into plants (e.g., with or without heritable modifications to the genome), or to regulate the expression of endogenous genes in plant cells or whole plants.

[0278] In some embodiments, the CRISPR system can be used to identify, edit, and / or silence genes encoding specific proteins, such as allergen proteins (e.g., allergen proteins in peanut, soybean, lentil, pea, green bean, and mung bean). Detailed descriptions of how to identify, edit, and / or silence genes encoding proteins are described, for example, in Nicolaou et al., "Molecular diagnosis of peanut and legume allergy", Curr. Opin. Allergy Clin. Immunol. 11(3):222-8, 2011, and WO2016205764 A1, both of which are incorporated herein by reference in their entirety.

[0279] Gene Drive A gene drive is a phenomenon in which the inheritance of a particular gene or set of genes is biased in favor of the other. A gene drive can be constructed using the CRISPR system described herein. For example, a CRISPR system can be designed to target and destroy a particular allele of a gene, which allows the cell to copy the second allele to fix the sequence. Due to the copying, the first allele is converted to the second allele, increasing the likelihood that the second allele will be transmitted to progeny. Detailed methods on how to construct a gene drive using the CRISPR system described herein are described, for example, in Hammond et al., "A CRISPR-Cas9 Gene Drive." Gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae”, Nat. Biotechnol. 34(1):78-83, 2016, which is incorporated herein by reference in its entirety.

[0280] Pooled Screening As described herein, pooled CRISPR screens are powerful tools for identifying genes involved in biological mechanisms, such as cell proliferation, drug resistance, and viral infection. The pooled CRISPR screen is a tool. Cells are transduced in large quantities with a library of guide RNA (gRNA)-encoding vectors described herein, and the distribution of gRNAs is measured before and after selective challenge. Pooled CRISPR screens work well for mechanisms that affect cell survival and proliferation, and can be extended to measure the activity of individual genes (e.g., using engineered reporter cell lines). Arrayed CRISPR screens, in which only one gene is targeted at a time, allow RNAseq to be used as a readout. In some embodiments, the CRISPR system described herein can be used for single-cell CRISPR screens. A detailed description of pooled CRISPR screening can be found, for example, 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.

[0281] Saturation mutagenesis (bashing) The CRISPR system described herein can be used for in situ saturation mutagenesis. In some embodiments, a pooled guide RNA library can be used to perform in situ saturation mutagenesis for specific genes or regulatory elements. Such methods can reveal important minimal features and distinct vulnerabilities of these genes or regulatory elements (e.g. enhancers). The methods are described, for example, in Canver et al. et al., “BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis”, Nature 527(7577):192-7, 2015, which is incorporated by reference in its entirety.

[0282] RNA-related Applications The CRISPR system described herein may have various RNA-related applications, such as regulating gene expression, degrading RNA molecules, inhibiting RNA expression, screening RNA or RNA products, determining the function 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. A detailed description of these applications can be found, for example, in WO 2016 / 205764 A1, which is incorporated herein by reference in its entirety. In various embodiments, the methods described herein may be performed in vitro, in vivo, or ex vivo.

[0283] For example, the CRISPR system described herein can be administered to a subject with a disease or disorder to target and induce cell death in diseased cells (e.g., cancer cells or cells infected with an infectious agent). For example, in some embodiments, the CRISPR system described herein can be used to target and induce cell death in cancer cells, the cancer cells being derived from a subject with Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, uterine cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or bladder cancer.

[0284] Regulating gene expression The CRISPR system described herein can be used to regulate gene expression. The CRISPR system can be used with suitable guide RNA to target gene expression through the control of RNA processing. Examples of the control of RNA processing can include RNA processing reactions, such as RNA splicing (e.g., alternative splicing), viral replication, and tRNA biogenesis. RNA targeting proteins can also be used in combination with suitable guide RNA to control RNA activation (RNAa). RNA activation is a small RNA guide and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNA (dsRNA) induces target gene expression at the transcriptional / epigenetic level. Since RNAa leads to the promotion of gene expression, the control of gene expression can be achieved by disrupting or lowering RNAa. In some embodiments, the method includes using RNA-targeting CRISPR as an alternative to, for example, interfering ribonucleic acid (e.g., siRNA, shRNA, or dsRNA). Methods for regulating gene expression are described, for example, in WO2016205764, which is incorporated by reference in its entirety.

[0285] Regulating RNA interference The control of interfering RNA or microRNA (miRNA) can help reduce off-target effects by shortening the life span of interfering RNA or miRNA in vivo or in vitro.In some embodiments, the target RNA can include interfering RNA, i.e., RNA involved in RNA interference pathway, such as small hairpin RNA (shRNA), small interfering RNA (siRNA), etc.In some embodiments, the example of the target RNA includes miRNA or double-stranded RNA (dsRNA).

[0286] In some embodiments, the RNA targeting protein and the appropriate guide RNA, if expressed selectively (e.g., spatially or temporally under the control of a regulatable promoter, such as a tissue or cell cycle specific promoter and / or enhancer), can be used to protect a cell or system (in vivo or in vitro) from RNA interference (RNAi) in said cell. This can be useful in adjacent tissues or cells where RNAi is not required, or for comparison purposes in cells or tissues where CRISPR-associated proteins and the appropriate crRNA are expressed and not expressed (i.e., RNAi is not and is controlled, respectively). The RNA targeting protein can be used to control or bind to a molecule that comprises or consists of RNA, such as a ribozyme, ribosome, or riboswitch. In some embodiments, the guide RNA can recruit the RNA targeting protein to the molecule so that the RNA targeting protein can bind to the molecule. Such methods are described, for example, in WO2016205764 and WO2017070605, both of which are incorporated by reference in their entireties.

[0287] Modifying riboswitches to control metabolic regulation Riboswitches are regulatory segments of messenger RNA that bind small molecules and then regulate gene expression. This mechanism allows cells to sense the intracellular concentration of the small molecule. A particular riboswitch typically regulates adjacent genes by altering the transcription, translation, or splicing of the gene. Thus, in some embodiments, riboswitch activity can be controlled by using an RNA targeting protein in combination with a suitable guide RNA to target the riboswitch. This can be achieved through cleavage of the riboswitch or binding to the riboswitch. Methods of regulating riboswitches using the CRISPR system are described, for example, in WO2016205764 and WO2017070605, both of which are incorporated herein by reference in their entirety.

[0288] RNA modification In some embodiments, 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 used to modify an RNA sequence (e.g., mRNA). In some embodiments, the CRISPR-associated protein contains one or more mutations (e.g., in the catalytic domain) that prevent the CRISPR-associated protein from cleaving RNA.

[0289] In some embodiments, the CRISPR-associated protein can be used in an RNA-binding fusion polypeptide that includes a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID) fused to an RNA-binding domain, such as MS2 (aka MS2 coat protein), Qbeta (aka Qbeta coat protein), or PP7 (aka PP7 coat protein). The amino acid sequences of the RNA-binding domains MS2, Qbeta, and PP7 are set forth below: MS2 (MS2 coat protein) MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY Qbeta (Qbeta coat protein) MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY PP7 (PP7 coat protein) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVVQATSEDLVVNLVPLGR

[0290] In some embodiments, the RNA-binding domain can recruit the RNA-binding fusion polypeptide (which has a base-editing domain) to an effector complex by binding to a specific sequence (e.g., an aptamer sequence) or secondary structure motif on the crRNA of the systems described herein (e.g., when the crRNA is in an effector-crRNA complex). For example, in some embodiments, the CRISPR system includes a CRISPR-associated protein, a crRNA with an aptamer sequence (e.g., an MS2-binding loop, a QBeta-binding loop, or a PP7-binding loop), and an RNA-binding fusion polypeptide with a base-editing domain fused to an RNA-binding domain that specifically binds to the aptamer sequence. In this system, the CRISPR-associated protein forms a complex with the crRNA with the aptamer sequence. Furthermore, the RNA-binding fusion polypeptide forms a tripartite complex that can modify the target RNA by binding to the crRNA (via the aptamer sequence).

[0291] Methods of using the CRISPR system for base editing are described, for example, in WO 2017 / 219027, which is incorporated by reference in its entirety, particularly with respect to its discussion of RNA modification.

[0292] RNA splicing In some embodiments, the inactivated CRISPR-associated proteins described herein (e.g., CRISPR-associated proteins with one or more mutations in catalytic domain) can be used to target and bind to specific splicing sites on RNA transcripts. Binding of inactivated CRISPR-associated proteins to RNA conformationally inhibits spliceosome interaction with transcripts, allowing modification of the frequency of generation of specific transcript isoforms. Such methods can be used to treat disease by exon skipping, such that exons with mutations can be skipped in mature proteins. Methods of modifying splicing using CRISPR systems are described, for example, in WO 2017 / 219027, which is incorporated herein by reference in its entirety, particularly with respect to the discussion of RNA splicing.

[0293] therapeutic use The CRISPR systems described herein may have a variety of therapeutic applications, which may be based on one or more of the following capabilities of the subject CRISPR / Cas13e or Cas13f systems, both in vitro and in vivo: the ability to induce cellular senescence, induce cell cycle arrest, inhibit cell growth and / or proliferation, induce apoptosis, induce necrosis, and others.

[0294] In some embodiments, the new CRISPR system can be used to treat a variety of diseases and disorders, such as genetic disorders (e.g., monogenic diseases), diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne muscular dystrophy (DMD), BCL11a targeting), various cancers, and others.

[0295] In some embodiments, the CRISPR system described herein can be used to edit a target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleic acid residues) to modify the target nucleic acid. For example, in some embodiments, the CRISPR system described herein includes an exogenous donor template nucleic acid (e.g., a DNA molecule or an RNA molecule) that includes a desired nucleic acid sequence. Resolution of the cleavage event induced by the CRISPR system described herein causes the cell's molecular machinery to utilize the exogenous donor template nucleic acid to repair and / or resolve the cleavage event. Alternatively, the cell's molecular machinery can utilize an endogenous template to repair and / or resolve the cleavage event. In some embodiments, the CRISPR system described herein can be used to modify the target nucleic acid to create an insertion, deletion, and / or point mutation. In some embodiments, the insertion is a scarless insertion (i.e., the insertion of the intended nucleic acid sequence into the target nucleic acid without the resolution of the cleavage event resulting in additional unintended nucleic acid sequence). The donor template nucleic acid can be a double-stranded or single-stranded nucleic acid molecule (e.g., DNA or RNA). Methods for designing exogenous donor template nucleic acids are described, for example, in WO 2016 / 094874 A1, the entire contents of which are expressly incorporated herein by reference.

[0296] In one aspect, the CRISPR system described herein can be used to treat diseases (e.g., splicing defects or truncations) caused by overexpression of RNA, toxic RNA, and / or mutant RNA. For example, expression of toxic RNA can be associated with the formation of nuclear inclusions and late-onset degenerative changes in the brain, heart, or skeletal muscle. In some embodiments, the disorder is myotonic dystrophy. In myotonic dystrophy, the primary pathogenic effect of toxic RNA is to sequester binding proteins and impair the regulation of alternative splicing (see, e.g., Osborne et al., "RNA-dominant diseases", Hum. Mol. Genet., 2009 Apr. 15; 18(8): 1471-81). Myotonic dystrophy (DM) presents a particularly challenging disease for geneticists, as it results in a very wide range of clinical features. It has been noted that the classical form of DM, now called DM type 1 (DM1), is caused by an expansion of CTG repeats in the 3' untranslated region (UTR) of DMPK, a gene that encodes a cytosolic protein kinase. The CRISPR system described herein can target overexpressed or toxic RNA, such as the DMPK gene, or any misregulated alternative splicing in DM1 skeletal muscle, heart, or brain.

[0297] Additionally, the CRISPR system described herein can target trans-acting mutations affecting RNA-dependent functions that cause various diseases, such as Prader-Willi syndrome, spinal muscular atrophy (SMA), and dyskeratosis congenita. A list of diseases that can be treated using the CRISPR system described herein is summarized in Cooper et al., "RNA and disease", Cell, 136.4 (2009): 777-793, and WO 2016 / 205764 A1, both of which are incorporated herein by reference in their entirety. One of ordinary skill in the art will understand how to use the new CRISPR system to treat these diseases.

[0298] The CRISPR system described herein can also be used to treat a variety of tauopathies, including, for example, primary and secondary tauopathies, such as primary age-related tauopathy (PART) / neurofibrillary tangles (NFT)-predominant senile dementia (NFTs similar to those seen in Alzheimer's disease (AD), but without plaques), dementia pugilistica (chronic traumatic encephalopathy), and progressive supranuclear palsy. A useful list of tauopathies and methods of treating these diseases are described, for example, in International Publication No. WO2016205764, which is incorporated herein by reference in its entirety.

[0299] Also, the CRISPR system described herein can be used to target mutations that disrupt cis-acting splicing code, which can cause splicing defects and diseases.Examples of these diseases include motor neuron degenerative diseases (e.g. spinal muscular atrophy) resulting from deletion of SMN1 gene, Duchenne muscular dystrophy (DMD), frontotemporal dementia, Parkinsonism associated with chromosome 17 (FTDP-17), and cystic fibrosis.

[0300] The CRISPR system described herein can also be used for antiviral activity, particularly against RNA viruses.CRISPR-associated proteins can target viral RNA using appropriate guide RNAs selected to target viral RNA sequences.

[0301] The CRISPR system described herein can also be used to treat cancer in a subject (e.g., a human subject). For example, the CRISPR-associated proteins described herein can be programmed with crRNAs that target RNA molecules that are abnormal (e.g., contain point mutations or are alternatively spliced) and found in cancer cells to induce cell death (e.g., via apoptosis) in cancer cells.

[0302] The CRISPR system described herein can also be used to treat autoimmune diseases or disorders in a subject (e.g., a human subject). For example, the CRISPR-associated proteins described herein can be programmed with crRNAs that target RNA molecules found in cells that are abnormal (e.g., contain point mutations or are alternatively spliced) and responsible for causing an autoimmune disease or disorder.

[0303] In addition, the CRISPR system described herein can also be used to treat infectious diseases in subjects. For example, the CRISPR-associated proteins described herein can be programmed with crRNAs that target RNA molecules expressed by infectious agents (e.g., bacteria, viruses, parasites, or protozoa) to target infectious agent cells and induce cell death in the infectious agent cells. The CRISPR system can also be used to treat diseases in which intracellular infectious agents infect the cells of a host subject. By programming the CRISPR-associated proteins to target RNA molecules encoded by infectious agent genes, cells infected with infectious agents can be targeted to induce cell death.

[0304] Furthermore, in vitro RNA detection assays can be used to detect specific RNA substrates. CRISPR-associated proteins can be used for RNA-based detection in living cells. Examples of applications include, for example, disease-specific RNA detection in diagnostics.

[0305] Detailed descriptions of the therapeutic applications of the CRISPR systems described herein can be found, for example, in U.S. Pat. No. 8,795,965, EP 3009511, WO 2016205764, and WO 2017070605, each of which is incorporated by reference in its entirety.

[0306] Cells and their progeny In certain embodiments, the methods of the invention can be used to introduce a CRISPR system described herein into a cell to alter the production of one or more cellular products in the cell and / or its progeny, such as an antibody, starch, ethanol, or any other desired product. Such cells and their progeny are within the scope of the invention.

[0307] In certain embodiments, the methods and / or CRISPR systems described herein lead to the modification of the translation and / or transcription of one or more RNA products of a cell. For example, the modification can lead to an increase in the transcription / translation / expression of the RNA product. In other embodiments, the modification can lead to a decrease in the transcription / translation / expression of the RNA product.

[0308] In certain embodiments, the cell is a prokaryotic cell.

[0309] In certain embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, including a human cell (primary human cell or established human cell line). In certain embodiments, the cell is a non-human mammalian cell, e.g., a cell from a non-human primate (e.g., monkey), cow, sheep, goat, pig, horse, dog, cat, rodent (e.g., rabbit, mouse, rat, hamster, etc.). In certain embodiments, the cell is from a fish (e.g., salmon), bird (e.g., poultry including chicks, ducks, geese), reptile, crustacean (e.g., oyster, clam, lobster, shrimp), insect, worm, yeast, etc. In certain embodiments, the cell is from a plant, e.g., a monocotyledonous or dicotyledonous plant. In certain embodiments, the plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, legumes, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, sugar beet, sunflower, and wheat. In certain embodiments, the plant is a cereal crop (barley, maize, millet, rice, rye, sorghum, and wheat). In certain embodiments, the plant is a tuber (cassava and potato). In certain embodiments, the plant is a sugar crop (sugar beet and sugarcane). In certain embodiments, the plant is an oil crop (soybean, groundnut or peanut, rapeseed or canola, sunflower, and oil palm fruit). In certain embodiments, the plant is a fiber crop (cotton). In certain embodiments, the plant is The plant may be a tree (such as a peach or nectarine tree, an apple or pear tree, a nut tree such as an almond, walnut, or pistachio tree, or a citrus tree such as an orange, grapefruit, or lemon tree), a grass, a vegetable, a fruit, or an algae. In certain embodiments, the plant is a Solanaceae plant; a Brassica plant; a Lactuca plant; a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0310] A related aspect provides a cell, or its progeny, modified by the methods of the invention using the CRISPR system described herein.

[0311] In certain embodiments, the cells are modified in vitro, in vivo, or ex vivo.

[0312] In certain embodiments, the cells are stem cells.

[0313] 7.Delivery With this disclosure and knowledge in the art, the CRISPR system described herein, or any of its components described herein (Cas proteins, derivatives, functional fragments, or various fusions or adducts, and guide RNA / crRNA), its nucleic acid molecules, and / or nucleic acid molecules encoding or providing its components, can be delivered by various delivery systems, such as vectors, e.g., plasmid vectors and viral delivery vectors, using any means suitable in the art, including but not limited to electroporation, lipofection, microinjection, transfection, sonication, gene guns, and others.

[0314] In certain embodiments, the CRISPR-associated proteins and / or any RNA (e.g., guide RNA or crRNA) and / or accessory proteins can be delivered using a suitable vector, such as a plasmid or viral vector, such as adeno-associated virus (AAV), lentivirus, adenovirus, retrovirus vectors, and other viral vectors, or combinations thereof. The proteins and one or more crRNAs can be packaged in one or more vectors, such as a plasmid or viral vector. For bacterial applications, nucleic acids encoding any of the components of the CRISPR system described herein can be delivered to the bacteria using phages. Exemplary phages include, but are not limited to, T4 phage, Mu, lambda phage, T5 phage, T7 phage, T3 phage, Φ29, M13, MS2, Qβ, and ΦX174.

[0315] In some embodiments, the vector, e.g., a plasmid or viral vector, is delivered to the tissue of interest, e.g., by intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be via either a single dose or multiple doses. Those skilled in the art will appreciate that the actual dosage to be delivered herein may vary widely depending on a variety of factors, such as the choice of vector, the target cell, organism, tissue, the general condition of the subject to be treated, the degree of transformation / modification desired, the route of administration, the mode of administration, the type of transformation / modification desired, etc.

[0316] In certain embodiments, delivery is via adenovirus, which delivers at least 1×10 5 The adenovirus is delivered in a single dose containing 1000 particles (also called particle units, pu). In some embodiments, the dose is preferably at least about 1×10 6 Particles, at least about 1 x 10 7 Particles, at least about 1 x 10 8 Particles, at least about 1 x 10 9The adenovirus is a particle. The delivery method and dosage are described, for example, in WO 2016205764 No. 8,454,972 B2, both of which are incorporated herein by reference in their entireties.

[0317] In some embodiments, delivery is via a plasmid. The dosage may be a sufficient number of plasmids to elicit a response. In some cases, a suitable amount of plasmid DNA in a plasmid composition may be about 0.1 to about 2 mg. A plasmid will typically include: (i) a promoter; (ii) sequences encoding nucleic acid-targeted CRISPR-associated proteins and / or accessory proteins, each operably linked to a promoter (e.g., the same promoter or different promoters); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of (ii) and operably linked to (ii). The plasmid may also encode the RNA components of the CRISPR complex, although one or more of these may alternatively be encoded on a different vector. The frequency of administration is within the purview of a medical or veterinary practitioner (e.g., physician, veterinarian), or one of ordinary skill in the art.

[0318] In another embodiment, delivery may be arranged by methods known to those skilled in the art, such as via liposome or lipofection formulations. Such methods are described, for example, in WO2016205764, as well as 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.

[0319] In some embodiments, delivery is via nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful for delivering RNA.

[0320] Another way to introduce one or more components of the new CRISPR system into cells is by using a cell membrane-permeable peptide (CPP). In some embodiments, the cell membrane-permeable peptide is linked to a CRISPR-associated protein. In some embodiments, the CRISPR-associated protein and / or guide RNA is linked to one or more CPPs to be effectively delivered inside a cell (e.g., a plant protoplast). In some embodiments, the CRISPR-associated protein and / or guide RNA is encoded by one or more circular or non-circular DNA molecules that are linked to one or more CPPs for cellular delivery.

[0321] CPPs are short peptides of less than 35 amino acids derived from either proteins or chimeric sequences that can transport biomolecules across cell membranes independent of receptors. CPPs can be cationic peptides, peptides with hydrophobic sequences, amphipathic peptides, peptides with proline-rich and antimicrobial sequences, and chimeric or bipartite peptides. Examples of CPPs include Tat (a nuclear transcription activator protein required for viral replication by HIV type 1), penetratin, Kaposi's fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Arg sequence, guanine-rich-molecule transporter, and sweet arrow peptide. CPPs and methods of using them are described, for example, in Haellbrink et al., "Prediction of cell-penetrating peptides”,Methods Mol.Biol.,2015;1324:39-58;Ramakrishna et al.,“Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA”,Ge Nome Res., 2014 June;24(6):1020-7; and WO 2016205764 A1, each of which is incorporated by reference in its entirety.

[0322] Various delivery methods for the CRISPR systems described herein are also described, for example, in U.S. Pat. No. 8,795,965, EP 3009511, WO2016205764, and WO2017070605, each of which is incorporated by reference in its entirety.

[0323] 8. Kit Another aspect of the present invention provides kits comprising any two or more components of a subject CRISPR / Cas system described herein, such as Cas13e and Cas13f proteins, derivatives, functional fragments, or various fusions or adducts thereof, guideRNA / crRNA, complexes thereof, vectors embodying same, or hosts embodying same.

[0324] In certain embodiments, the kit further comprises instructions for use of the components contained therein and / or instructions for combining with additional components available elsewhere.

[0325] In certain embodiments, the kit further comprises one or more nucleotides, e.g., nucleotides corresponding to those useful for inserting a guide RNA coding sequence into a vector and operably linking the coding sequence to one or more control elements of the vector.

[0326] In certain embodiments, the kit further comprises one or more buffers that may be used to dissolve any of the components and / or provide suitable reaction conditions for one or more of the components. Such buffers may comprise one or more of PBS, HEPES, Tris, MOP, Na2CO3, NaHCO3, NaB, or combinations thereof. In certain embodiments, the reaction conditions comprise a suitable pH, e.g., a basic pH. In certain embodiments, the pH is between 7 and 10.

[0327] In certain embodiments, any one or more of the kit components may be stored in a suitable container. EXAMPLES

[0328] Example 1 Identification of novel Cas13e and Cas13f systems A computational pipeline was used to generate an extended database of class 2 CRISPR-Cas systems from genomic and metagenomic sources. Genomic and metagenomic sequences were downloaded from NCBI (Benson et al., 2013; Pruitt et al., 2012), NCBI whole genome sequencing (WGS), and DOE JGI Integrated Microbial Genomes (Markowitz et al., 2012). Proteins were predicted (Prodigal in anon mode (Hyatt et al., 2010)) in all contigs of at least 5 kb length and de-duplicated (i.e., removing identical protein sequences) to construct a complete protein database. Proteins larger than 600 residues were considered as large proteins (LPs). As the majority of currently identified Cas13 proteins are larger than 900 residues in size, to reduce the complexity of the calculations, only large proteins were further considered.

[0329] CRISPR arrays were run using Piler-CR (Ed. gar, PILER-CR: Fast and accurate identification of CRISPR repeats. BMC Bioinformatics 8:18, 2007). Non-overlapping large protein sequence-encoding ORFs located within ±10 kb from the CRISPR array were classified into CRISPR-proximal large protein-encoding clusters, and the encoded LPs were defined as Cas-LPs.

[0330] First, pairwise alignment between Cas-LPs was performed using BLASP to obtain BLASTP alignment results with Evalue<1E-10. Then, MCL was used to further cluster Cas-LPs based on the BLASTP results to generate families of Cas proteins.

[0331] Then, Cas-LP was aligned against all LPs using BLASTP to obtain BLASP alignment results with Evalue<1E-10. The Cas-LP family was further expanded according to the BLASTP alignment results. The Cas-LP families with less than twofold expansion after expansion were obtained for further analysis.

[0332] For functional characterization of the candidate Cas proteins, they were annotated using the protein family database Pfam (Finn et al., 2014), the NR database, and Cas proteins in NCBI. Multiple sequence alignments were then performed for each candidate Cas effector protein using MAFFT (Katoh and Standley, 2013). Conserved regions within these proteins were then analyzed using JPred and HHpred to identify candidate Cas proteins / families with two conserved RXXXXH motifs.

[0333] This analysis led to the identification of seven novel Cas13 effector proteins that fall into two new Cas13 families distinct from all previously identified Class 2 CRISPR-Cas systems, including the new Cas13e family, Cas13e.1 (SEQ ID NO:1) and Cas13e.2 (SEQ ID NO:2), and the new Cas13f family, Cas13f.1 (SEQ ID NO:3), Cas13f.2 (SEQ ID NO:4), Cas13f.3 (SEQ ID NO:5), Cas13f.4 (SEQ ID NO:6), and Cas13f.5 (SEQ ID NO:7). [ka] [ka] [ka]

[0334] DNA encoding the corresponding direct repeat (DR) sequence in each pre-crRNA sequence is SEQ ID NO: 8 to 14, respectively. GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC (SEQ ID NO: 8) GCTGAAGAAGCCTCCGATTTGAGAGGTGATTACAGC (SEQ ID NO: 9) GCTGTGATAGACCTCGATTTGTGGGGTAGTAACAGC (SEQ ID NO: 10) GCTGTGATAGACCTCGATTTGTGGGGTAGTAACAGC (SEQ ID NO: 11) GCTGTGATAGACCTCGATTTGTGGGGTAGTAACAGC (SEQ ID NO: 12) GCTGTGATGGGCCTCAATTTGTGGGGAAGTAACAGC (SEQ ID NO: 13) GCTGTGATAGGCCTCGATTTGTGGGGTAGTAACAGC (SEQ ID NO: 14)

[0335] The native (wild-type) DNA coding sequences for Cas13e.1, Cas13e.2, Cas13f.1, Cas13f.2, Cas13f.3, Cas13f.4, and Cas13f.5 proteins are SEQ ID NOs: 15 to 21, respectively. [ka] [ka] [ka] [ka] [ka]

[0336] The human codon-optimized coding sequences of the seven Cas13e and Cas13f proteins (i.e., Cas13e.1, Cas13e.2, Cas13f.1, Cas13f.2, Cas13f.3, Cas13f.4, and Cas13f.5) generated for further functional experiments are SEQ ID NOs: 22 to 28, respectively. [ka] [ka] [ka] [ka] [ka]

[0337] The seven CRISPR / Cas13e and Cas13f locus structures are shown in Figure 1.

[0338] Further analysis of the RNA secondary structure of the seven DR sequences within the pre-crRNA was performed using RNAfold, and the results are shown in Figure 2. It is clear that all shared a highly conserved secondary structure.

[0339] For example, in the Cas13e family, each DR sequence forms a secondary structure consisting of a 4 base pair stem (5'-GCUG-3') followed by a symmetric bulge of 5+5 nucleotides (excluding 4 stem nucleotides), followed by a 5 base pair stem (5'-GCC C / U C-3') and a terminal 8 base loop (5'-CGAUUUGU-3', excluding 2 stem nucleotides).

[0340] Similarly, in the Cas13f family (with one exception (Cas13f.4) Each DR sequence forms a secondary structure consisting of a 5 base pair stem (5'-GCUGU3') followed by a nearly symmetrical bulge of 5+4 nucleotides (excluding 4 stem nucleotides), followed by a 6 base pair stem (5'A / G CCUCG3') and a terminal 5 base loop (5'AUUUG3', excluding 2 stem nucleotides). The only exception is the DR of Cas13f.4, where the second step is one base pair shorter and an additional 2 bases are added to the first bulge, forming a mostly symmetrical 6+5 bulge.

[0341] Multiple sequence alignment of Cas13e and Cas13f proteins and previously identified Cas13a, Cas13b, Cas13c, and Cas13d family proteins using MAFFT revealed that Cas13e and Cas13f proteins are relatively closest to Cas13b protein on the phylogenetic tree (Figure 3).

[0342] Furthermore, with respect to the location of the RXXXXH motif relative to the N- and C-termini of the Cas protein, the Cas13e and Cas13f proteins, and to a lesser extent the Cas13b protein, have the RXXXXH motif closer to the N- and C-termini than Cas13a, Cas13c, and Cas13d (see Figure 4).

[0343] We then used I-TASSER to predict the 3D structure of the Cas13e protein and visualized the predicted structure using PyMOL. The two RXXXXH motifs, located very close to the N- and C-termini of Cas13e.1, are very close in the 3D structure (Figure 5).

[0344] Example 2 Cas13e is an effector RNase To confirm that the newly identified Cas13e protein is a valid RNase that functions in the CRISPR / Cas system, the Cas13e.1 coding sequence was codon-optimized for human expression (SEQ ID NO: 22) and cloned into a first plasmid carrying a GFP gene. Meanwhile, the coding sequence of a guide RNA (gRNA) targeting a reporter gene (mCherry) mRNA was cloned into a second plasmid carrying a GFP gene. The gRNA consists of a spacer coding region flanked by two direct repeat sequences for Cas13e.1 (SEQ ID NO: 29). The sequences of the GFP and mCherry reporter genes are SEQ ID NOs: 30-31, respectively. [ka]

[0345] HEK293T cells were cultured in 24-well tissue culture plates according to standard protocols and used for triple plasmid transfection using LIPOFECTAMINE® 3000 and P3000™ reagents to introduce three plasmids encoding Cas13e.1 protein, mCherry-targeting gRNA, and mCherry coding sequence, respectively. In negative control experiments, instead of using a plasmid encoding mCherry-targeting gRNA, a control plasmid encoding a non-targeting gRNA was used. Since GFP coding sequence is present in Cas13e.1 and gRNA plasmids, expression of GFP can be used as an internal control for transfection success / efficiency. See schematic diagram in Figure 6. Transfected HEK293T cells were then incubated at 37°C under 5% CO2 for approximately 24 hours before cells were examined under a fluorescent microscope.

[0346] As shown in Figure 7, cells transfected with mCherry-targeting gRNA and cells transfected with control non-targeting (NT) gRNA had equivalent growth and morphology in bright field microscopy, and GFP expression in both was largely equivalent. However, the RFP signal from mCherry expression was dramatically reduced by up to 75% based on flow cytometry analysis (Figure 8). This suggests that Cas13e can utilize mCherry-targeting gRNA to efficiently knock down mCherry mRNA levels, and consequently mCherry protein expression.

[0347] Example 3 Effective orientation of sgRNA for Cas13e Because the Cas13e system can theoretically utilize either the DR+spacer (5'DR) or spacer+DR (3'DR) orientation, this experiment was designed to determine which is the exact orientation utilized by Cas13e.

[0348] Using a triple transfection experimental setup similar to Example 2, only the 3'DR orientation (spacer+DR) was found to support significant mCherry knockdown, demonstrating that Cas13e utilizes a crRNA with a DR sequence at the 3' end of the spacer. See Figure 9.

[0349] The DR+spacer (5'DR) and spacer+DR (3'DR) SgRNAs are SEQ ID NOs: 32 and 33, respectively. GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGCGGTCTTCGATATTCAAGCGTCGGAAGACCT (SEQ ID NO: 32) GGTCTTCGATATTCAAGCGTCGGAAGACCTGCTGGAGCAGCCCCGATTTGTGGGGTGATTACAGC (SEQ ID NO: 33)

[0350] Example 4 Effect of spacer sequence length on specific and collateral activity of Cas13e.1 To study the effect of spacer sequence length on the specific and collateral activity of Cas13e.1, a set of sgRNAs targeting the mCherry reporter gene were designed with spacer sequence lengths of 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt (sequence numbers 34 to 40). TTGGTGCCGCGCAGCTTCAC (SEQ ID NO: 34) TTGGTGCCGCGCAGCTTCACCTTGT (SEQ ID NO: 35) TTGGTGCCGCGCAGCTTCACCTTGTAGATG (SEQ ID NO: 36) TTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTC (SEQ ID NO: 37) TTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGT (SEQ ID NO: 38) TTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGC (SEQ ID NO: 39) TTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGCAGGGA (SEQ ID NO: 40)

[0351] Using a triple transfection experimental setup similar to that in Example 2, the knockdown efficiency of mCherry and GFP genes was analyzed by flow cytometry.

[0352] The results of mCherry and GFP knockdown experiments showed the specific and non-specific (collateral) activities of Cas13e.1, respectively. It was found that Cas13e.1 has high specific activity with a spacer length of about 30 nt to about 50 nt. See Figure 10. Meanwhile, Cas13e.1 has the highest non-specific activity when the spacer length is about 30 nt. See Figure 11.

[0353] Example 5 Single-base RNA editing using dCas13e.1-ADAR2DD fusion To test whether Cas13e can be used for RNA single base editing, dCas13e.1 was generated by mutating two RXXXXH motifs to eliminate RNase activity. A high-fidelity ADAR2DD mutant with E488Q and T375G double mutations was then fused to dCas13e.1 (at the C-terminus) to generate a putative A to G single base RNA editor, named dCas13e.1-ADAR2DD. See coding sequence in SEQ ID NO:41. [ka]

[0354] For use as a putative RNA base-editor target, the wild-type mCherry coding sequence was mutated to create a premature stop codon TAG (see bold double underlined sequence in SEQ ID NO: 42) such that functional mCherry protein would not be produced without the A to G correction by the RNA base editor. See Figures 12 and 14. A gRNA was then designed to create the desired A to G edit (Figures 12 and 14), and the CX530 plasmid encoding the dCas13e.1-ADAR2DD base editor, the CX537 / Cx538 plasmid encoding the sgRNA, and the CX337 plasmid encoding the mutated mCherry gene were triple-transfected into HEK293T cells using standard protocols. The transfected HEK293T cells were incubated at 37°C under 5% CO2 for 24 hours, and then the cells were subjected to flow cytometry to identify cells with corrected mCherry mRNA and expressing mCherry protein. See explanatory diagram in Figure 12. The results of flow cytometry analysis are shown in Figure 13.

[0355] It is clear that both gRNA-1 (SEQ ID NO:43) and gRNA-2 (SEQ ID NO:44) successfully correct the TAG premature stop codon to produce functional mCherry protein. [ka]

[0356] Example 6 Single-base RNA editing using short dCas13e.1-ADAR2DD fusions To determine the minimal size of dCas13e.1 that can be used for RNA single base editing, a series of five constructs were generated expressing progressively larger C-terminal deletions of dCas13e.1, each with less than 30 residues from the C-terminus (i.e., 30-, 60-, 90-, 120-, and 150-residue deletions). The resulting constructs were used to generate the coding sequence of dCas13e.1 fused to high-fidelity adar2 (ADAR2DD) at each C-terminus. These constructs were cloned into Vysz15 ("V15") to Vysz-19 ("V19") plasmids (FIG. 15) used for experiments similar to those in Example 4. In all these constructs, the fusion proteins were expressed from a CMV promoter (pCMV) and an enhancer (eCMV) immediately downstream of an intron to further enhance protein expression. Two nuclear localization signals (NLS) were placed at the N- and C-termini of the dCas13e.1 portion of the fusion, and the ADAR2 domain (e.g., ADAR2DD) was fused to the C-terminal NLS via an NLS linker and tagged at the C-terminus with an HA-tag. An EGFP coding sequence under the independent control of the EFS promoter (pEFS) was present downstream of the polyA addition sequence in all plasmids.

[0357] Interestingly, progressive C-terminal deletions were found to increase RNA base editing activity in the fusion editors such that the editor with a 150 C-terminal residue deletion (in V19) exhibited the highest base editing activity. See Figure 16. However, deletion of the 180-residue from the C-terminus appeared to abolish base editing activity. This suggests that the maximum / optimal deletion from the C-terminus of Cas13e.1 is likely to be between 150 and 180 residues.

[0358] Based on this finding, a series of N-terminal deletion mutants were generated for dCas13e.1 with 150 C-terminal residue deletions. Seven such N-terminal deletion mutants were generated with 30-, 60-, 90-, 120-, 150-, 180-, and 210-residue deletions, respectively. See FIG. 17. The results in FIG. 18 show that the best RNA editing activity was observed with 180 N-terminal residue deletions and 150 C-terminal residue deletions, i.e., a total of 330-residue deletions from the 775-residue Cas13e.1 protein (445-residue deletion optimal for generating ADAR2DD fusions). We showed that this was observed in a mutant carrying the dCas13e.1 base.

[0359] Example 7 Comparison of mammalian endogenous mRNA knockdown efficiency using various Cas13 proteins This experiment demonstrated that the Cas13e and Cas13f proteins, especially Cas13f.1, are highly effective at knocking down endogenous mammalian target mRNAs more efficiently than previously identified Cas13 proteins.

[0360] Specifically, five plasmids were constructed, each expressing one of the Cas13 proteins, namely, Cas13e.1 (SEQ ID NO: 22), Cas13f.1 (SEQ ID NO: 23), LwaCas13a (SEQ ID NO: 45), PspCas13b (SEQ ID NO: 46), and RxCas13d (SEQ ID NO: 47). Each plasmid also encoded an mCherry reporter gene and an sgRNA / crRNA coding sequence for each Cas13 protein flanked by two native DR sequences. These sgRNAs were designed to have a spacer sequence that targets ANXA4 mRNA. See SEQ ID NOs: 48-50. As negative controls, five additional plasmids were constructed, each encoding a non-targeting sgRNA / crRNA instead of the ANXA4-targeting sgRNA / crRNA ("Control NT Construct"). See FIG. 19. [ka] [ka] [ka]

[0361] The five Cas13 / sgRNA-encoding plasmids were transfected into HEK293 cells as in Example 4. After 24 hours of culture, cells expressing mCherry were isolated by flow cytometry and ANXA4 mRNA expression was determined using RT-PCR to assess knockdown efficiency compared to control cells transfected with Cas13 / NT-encoding plasmids.

[0362] Figure 20 shows that Cas13b alone provided marginal ANXA4 mRNA knockdown, while Cas13e.1, Cas13f.1, and Cas13d each provided greater than 80% knockdown of the target ANXA4 mRNA. Notably, Cas13e.1 appeared to have the most robust knockdown efficiency.

Claims

1. (1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA and a direct repeat (DR) sequence 3' to the spacer sequence; and (2) A CRISPR-associated protein (Cas) having any one of the amino acid sequences of SEQ ID NOs: 1 to 7, or a derivative or functional fragment of the Cas. A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex comprising: The Cas, the derivatives and the functional fragments of the Cas are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA, A CRISPR-Cas complex, with the proviso that the spacer sequence is not 100% complementary to a naturally occurring bacteriophage nucleic acid when the complex comprises the Cas of any one of SEQ ID NOs: 1-7.

2. The CRISPR-Cas complex of claim 1, wherein the DR sequence has substantially the same secondary structure as any one of SEQ ID NOs: 8-14.

3. The CRISPR-Cas complex of claim 1, wherein the DR sequence is encoded by any one of SEQ ID NOs: 8-14.

4. The CRISPR-Cas complex of any one of claims 1 to 3, wherein the target RNA is encoded by eukaryotic DNA.

5. 5. The CRISPR-Cas complex of claim 4, wherein the eukaryotic DNA is non-human mammalian DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, avian DNA, reptile DNA, rodent DNA, fish DNA, worm / nematode DNA, yeast DNA.

6. The CRISPR-Cas complex of any one of claims 1 to 5, wherein the target RNA is mRNA.

7. 7. The CRISPR-Cas complex of any one of claims 1 to 6, wherein the spacer sequence is 15 to 60 nucleotides, 25 to 50 nucleotides, or about 30 nucleotides.

8. The CRISPR-Cas complex of any one of claims 1 to 7, wherein the spacer sequence is 90-100% complementary to the target RNA.

9. The CRISPR-Cas complex of any one of claims 1 to 8, wherein the derivative comprises a conservative amino acid substitution of one or more residues of any one of SEQ ID NOs: 1 to 7.

10. The CRISPR-Cas complex of claim 9, wherein the derivative comprises only conservative amino acid substitutions.

11. The CRISPR-Cas complex of any one of claims 1 to 10, wherein the derivative has an identical sequence within the HEPN domain or RXXXXH motif to a wild-type Cas of any one of SEQ ID NOs: 1 to 7.

12. The derivative is capable of binding to the RNA guide sequence that hybridizes to the target RNA, but is unable to bind to the RNA guide sequence due to a mutation in the RNase catalytic site of the Cas. The CRISPR-Cas complex according to any one of claims 1 to 9, which has no activity.

13. The CRISPR-Cas complex of claim 12, wherein the derivative has an N-terminal deletion of up to 210 residues and / or a C-terminal deletion of up to 180 residues.

14. The CRISPR-Cas complex of claim 13, wherein the derivative has an N-terminal deletion of about 180 residues and / or a C-terminal deletion of about 150 residues.

15. The CRISPR-Cas complex of any one of claims 12 to 14, wherein the derivative further comprises an RNA base-editing domain.

16. 16. The CRISPR-Cas complex of claim 15, wherein the RNA base-editing domain is an adenosine deaminase, such as a double-stranded RNA-specific adenosine deaminase (e.g., ADAR1 or ADAR2); apolipoprotein B mRNA editing enzyme; catalytic polypeptide-like (APOBEC); or activation-induced cytidine deaminase (AID).

17. 17. The CRISPR-Cas complex of claim 16, wherein the ADAR2 has an E488Q / T375G double mutation or is ADAR2DD.

18. The CRISPR-Cas complex of any one of claims 15 to 17, wherein the base-editing domain is further fused to an RNA binding domain, such as MS2.

19. The CRISPR-Cas complex of any one of claims 12 to 14, wherein the derivative further comprises an RNA methyltransferase, an RNA demethylase, an RNA splicing modifier, a localization factor, or a translation modifier.

20. The CRISPR-Cas complex of any one of claims 1 to 19, wherein the Cas, derivative, or functional fragment comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).

21. The CRISPR-Cas complex of any one of claims 1 to 20, wherein targeting of the target RNA results in modification of the target RNA.

22. 22. The CRISPR-Cas complex of claim 21, wherein the modification of the target RNA is cleavage of the target RNA.

23. 22. The CRISPR-Cas complex of claim 21, wherein the modification of the target RNA is deamination of adenosine (A) to inosine (I).

24. The CRISPR-Cas complex of any one of claims 1 to 23, further comprising a target RNA comprising a sequence capable of hybridizing to the spacer sequence.

25. (1) A fusion protein comprising a Cas, a derivative thereof, or a functional fragment thereof according to any one of claims 1 to 24, and (2) a heterologous functional domain.

26. The heterologous functional domain may be: a nuclear localization signal (NLS), a reporter protein or a detection label (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), a localization signal, a protein targeting moiety, a DNA binding domain ( 26. The fusion protein of claim 25, comprising a polypeptide having an ssRNA cleavage activity, a polypeptide having dsRNA cleavage activity, a polypeptide having ssDNA cleavage activity, a polypeptide having dsDNA cleavage activity, a DNA or RNA ligase, or any combination thereof.

27. 27. The fusion protein of claim 25 or 26, wherein the heterologous functional domain is fused N-terminally, C-terminally, or internally within the fusion protein.

28. (2) A conjugate comprising (1) a Cas, a derivative thereof, or a functional fragment thereof described in any one of claims 1 to 24, conjugated to a heterologous functional moiety.

29. The heterologous functional moiety may be: a nuclear localization signal (NLS), a reporter protein or detection label (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), a localization signal, a protein targeting moiety, a DNA binding domain (e.g., MBP, Lex A DBD, Gal4 29. The conjugate of claim 28, comprising a polypeptide having a ssRNA cleavage activity, a polypeptide having dsRNA cleavage activity, a polypeptide having ssDNA cleavage activity, a polypeptide having dsDNA cleavage activity, a DNA or RNA ligase, or any combination thereof.

30. 30. The conjugate of claim 28 or 29, wherein the heterologous functional moiety is conjugated N-terminally, C-terminally, or internally to the Cas, the derivative, or the functional fragment thereof.

31. A polynucleotide encoding any one of SEQ ID NOs: 1 to 7, or a derivative thereof, or a functional fragment thereof, or a fusion protein thereof, provided that the polynucleotide is not any one of SEQ ID NOs: 15 to 21.

32. 32. The polynucleotide of claim 31, which is codon-optimized for expression in a cell.

33. The polynucleotide of claim 32, wherein the cell is a eukaryotic cell.

34. A non-naturally occurring polynucleotide comprising a derivative of any one of SEQ ID NOs:8-14, said derivative (i) having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotide additions, deletions, or substitutions compared to any one of SEQ ID NOs:8-14; (ii) having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 97% sequence identity to any one of SEQ ID NOs:8-14; (iii) hybridizing under stringent conditions to any one of SEQ ID NOs:8-14, or any of (i) and (ii); or (iv) being the complement of any of (i)-(iii), wherein said derivative is a non-naturally occurring polynucleotide comprising any one of SEQ ID NOs:8-14, 14, and said derivative encodes (or is) an RNA that maintains substantially the same secondary structure as any of the RNAs encoded by SEQ ID NOs: 8-14.

35. The non-naturally occurring polynucleotide of claim 34, wherein the derivative functions as a DR sequence of any one of the Cas, derivatives, or functional fragments thereof of any one of claims 1 to 24.

36. A vector comprising the polynucleotide according to any one of claims 31 to 35.

37. 37. The vector of claim 36, wherein the polynucleotide is operably linked to a promoter and optionally an enhancer.

38. 38. The vector of claim 37, wherein the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a tissue-specific promoter.

39. The vector according to any one of claims 36 to 38, which is a plasmid.

40. 39. The vector of any one of claims 36 to 38, which is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an AAV vector, or a lentiviral vector.

41. 41. The vector of claim 40, wherein the AAV vector is a recombinant AAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

42. A delivery system comprising: (1) a delivery vehicle; and (2) a CRISPR-Cas complex according to any one of claims 1 to 24, a fusion protein according to any one of claims 25 to 27, a conjugate according to any one of claims 28 to 30, a polynucleotide according to any one of claims 31 to 33, or a vector according to any one of claims 36 to 41.

43. 43. The delivery system of claim 42, wherein the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

44. A cell or its progeny comprising a CRISPR-Cas complex according to any one of claims 1 to 24, a fusion protein according to any one of claims 25 to 27, a conjugate according to any one of claims 28 to 30, a polynucleotide according to any one of claims 31 to 33, or a vector according to any one of claims 36 to 41.

45. 45. The cell or progeny thereof of claim 44, which 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 bacterial cell).

46. 46. ​​A non-human multicellular eukaryotic organism comprising a cell according to claim 44 or 45.

47. 47. The non-human multicellular eukaryote of claim 46, which is an animal (e.g., rodent or primate) model for a human genetic disorder.

48. A method for modifying a target RNA, comprising contacting the target RNA with a CRISPR-Cas complex according to any one of claims 1 to 24, wherein the spacer sequence is wherein said Cas, derivative or functional fragment binds to said RNA guide sequence to form said complex; said complex binds to said target RNA; and upon binding of said complex to said target RNA, said Cas, derivative or functional fragment modifies said target RNA.

49. 49. The method of Claim 48, wherein the target RNA is modified by cleavage by the Cas.

50. 49. The method of claim 48, wherein the target RNA is modified by deamination with a derivative comprising double-stranded RNA-specific adenosine deaminase.

51. 51. The method of any one of claims 48 to 50, wherein the target RNA is mRNA, tRNA, rRNA, non-coding RNA, lncRNA, or nuclear RNA.

52. The method of any one of claims 48 to 51, wherein upon binding of the complex to the target RNA, the Cas, the derivatives, and the functional fragments do not exhibit substantial (or detectable) collateral RNase activity.

53. The method of any one of claims 48 to 52, wherein the target RNA is intracellular.

54. 54. The method of claim 53, wherein the cell is a cancer cell.

55. 54. The method of claim 53, wherein the cell is infected with an infectious agent.

56. 56. The method of claim 55, wherein the infectious agent is a virus, a prion, a protozoan, a fungus, or a parasite.

57. 57. The method of any one of claims 53 to 56, wherein the CRISPR-Cas complex is encoded by a first polynucleotide encoding any one of SEQ ID NOs: 1 to 7, or a derivative or functional fragment thereof, and a second polynucleotide comprising any one of SEQ ID NOs: 8 to 14, and a sequence encoding a spacer RNA capable of binding to the target RNA, and the first polynucleotide and the second polynucleotide are introduced into the cell.

58. 58. The method of claim 57, wherein the first polynucleotide and the second polynucleotide are introduced into the cell by the same vector.

59. 59. The method of any one of claims 53 to 58, which 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 proliferation 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.

60. A method of treating a condition or disease in a subject in need of such treatment, comprising administering to the subject a composition comprising the CRISPR-Cas complex of any one of claims 1 to 24, or a polynucleotide encoding the same; the spacer sequence is complementary to at least 15 nucleotides of a target RNA associated with the condition or disease; the Cas, derivative, or functional fragment binds to the RNA guide sequence to form the complex; the complex binds to the target RNA; and upon binding of the complex to the target RNA, the Cas, derivative, or functional fragment cleaves the target RNA. treating said condition or disease in said subject by inhibiting

61. 61. The method of claim 60, wherein the condition or disease is cancer or an infectious disease.

62. 62. The method of claim 61, wherein the cancer is Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, uterine cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or bladder cancer.

63. 63. The method of any one of claims 60 to 62, which is an in vitro method, an in vivo method, or an ex vivo method.

64. 60. A cell or progeny thereof obtainable by a method according to any one of claims 48 to 59, said cell or progeny comprising a non-naturally occurring modification (e.g. a non-naturally occurring modification in the transcribed RNA of said cell / progeny).

65. 31. A method for detecting the presence of a target RNA, comprising contacting said target RNA with a composition comprising a fusion protein according to any one of claims 25 to 27, or a conjugate according to any one of claims 28 to 30, or a polynucleotide encoding said fusion protein, wherein said fusion protein or said conjugate comprises a detectable label (e.g. one that can be detected by fluorescence, Northern blot or FISH) and a synthetic spacer sequence capable of binding to said target RNA.

66. 1. A eukaryotic cell comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex, said CRISPR-Cas complex comprising: (1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA and a direct repeat (DR) sequence 3' to the spacer sequence; and (2) A CRISPR-associated protein (Cas) having an amino acid sequence of any one of SEQ ID NOs: 1 to 7, or a derivative or functional fragment of the Cas; A eukaryotic cell, wherein the Cas, the derivatives and the functional fragments of the Cas are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.

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