Compositions and methods for identifying the functional effects of RNA binding protein (RBP)-RNA interactions

Abstract

The present disclosure provides compositions and methods for identifying the functional effects / phenotypes associated with RNA binding protein (RBP)-RNA interactions (PRIs).

Description

Atty. Dkt. No.: 115872-3287COMPOSITIONS AND METHODS FOR IDENTIFYING THE FUNCTIONAL EFFECTS OF RNA BINDING PROTEIN (RBP)-RNA INTERACTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 680,804, filed August 8, 2024, the contents of which are incorporated herein by reference in its-entirety.TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for identifying the functional effects / phenotypes associated with RNA binding protein (RBP)-RNA interactions (PRIs).GOVERNMENT SUPPORT

[0003] This invention was made with government support under GM124909 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Site-specific interactions between RNA binding proteins (RBPs) and regulatory RNA sequences govern RNA stability, processing, and translation - processes essential to the function of many genes necessary for cancer cell survival and proliferation as well as pathogenic processes in many benign diseases. However, these interactions are poorly understood due to major methodologic limitations in identifying the physiological relevance of these interactions. Since RBP-RNA interactions (PRIs) are druggable in principle, the substantial limitations of current methods likely results in a massive underestimation of the druggable mutations in cancer and treatment targets in benign diseases.

[0006] Accordingly, there is an urgent need for compositions and methods for identifying the functional effects / phenotypes associated with various PRIs.-1-4856-2302-0756.1Atty. Dkt. No.: 115872-3287SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a fusion protein comprising doublestranded RNA binding protein (RBP) domain, a nuclease-defective Cast 3 domain, and one or more subcellular-localization sequences. In some embodiments, the double-stranded RBP domain is the double-stranded RBP domain of PKR, ADAR, Rnase III, or DICER. In some embodiments, the double-stranded RBP domain comprises the amino acid sequence of any of SEQ ID NO: 69 or SEQ ID NOs: 88-90. Additionally or alternatively, in some embodiments of the fusion protein disclosed herein, the nuclease-defective Casl3 domain is dCasl3a, dCasl3b, or dCasl3d. In certain embodiments, the nuclease-defective Cast 3 domain comprises the amino acid sequence of any of SEQ ID NOs: 1-3 or 87.

[0008] The one or more subcellular-localization sequences may be fused to the N- terminus or the C-terminus of the fusion protein. In some embodiments, the one or more subcellular-localization sequences may be fused to the N-terminus or the C-terminus of the PKR double-stranded RBP domain. Additionally or alternatively, in some embodiments, the one or more subcellular-localization sequences may be fused to the N-terminus or the C- terminus of the nuclease-defective Cast 3 domain. In some embodiments, the one or more subcellular-localization sequences are fused to any of the PKR double-stranded RBP domain, or the nuclease-defective Casl3 domain via one or more linkers. In other embodiments, the subcellular-localization sequences are fused to any of the PKR double-stranded RBP domain, or the nuclease-defective Cast 3 domain without a linker.

[0009] In any of the preceding embodiments of the fusion protein disclosed herein, the one or more subcellular-localization sequences are selected from the group consisting of nuclear localization sequences, Endoplasmic Reticulum membrane localization sequences, mitochondrial localization sequences, cytoplasmic localization sequences, nucleolus localization sequences, and plasma membrane localization sequences. Additionally or alternatively, in some embodiments, the one or more subcellular-localization sequences comprise the amino acid sequences of any of SEQ ID NOs: 4-16.

[0010] In certain embodiments, the fusion protein of the present technology comprises one subcellular-localization sequence located at the N-terminus of the nuclease-defective Cast 3 domain and / or the N-terminus of the PKR double-stranded RBP domain. In other-2-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 embodiments, the fusion protein of the present technology comprises one subcellular- localization sequence located at the C-terminus of the nuclease-defective Cast 3 domain and / or the C-terminus of the PKR double-stranded RBP domain.

[0011] Additionally or alternatively, in certain embodiments, the fusion protein of the present technology comprises at least two subcellular-localization sequences. The at least two subcellular-localization sequences may be identical or different. In some embodiments, the at least two subcellular-localization sequences are located at the N-terminus of the nuclease-defective Casl3 domain and / or the N-terminus of the PKR double-stranded RBP domain. In certain embodiments, the at least two subcellular-localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain and / or the C-terminus of the PKR double-stranded RBP domain.

[0012] In other embodiments, one of the at least two subcellular-localization sequences is located at the N-terminus of the nuclease-defective Cast 3 domain and the N-terminus of the PKR double-stranded RBP domain, and one of the at least two subcellular-localization sequences is located at the C-terminus of the nuclease-defective Cast 3 domain and the C- terminus of the PKR double-stranded RBP domain

[0013] Additionally or alternatively, in some embodiments, the fusion protein further comprises a protein tag. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags.

[0014] In any of the preceding embodiments, the fusion proteins of the present technology further comprise a detectable protein, such as a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof. Examples of bioluminescent protein include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent protein include, but are not limited to, P-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent protein include, but are-3-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 not limited to, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKatel, LSS-mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS- CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0015] Additionally or alternatively, in some embodiments, the PKR double-stranded RBP domain and / or the nuclease-defective Casl3 domain and the detectable protein are linked via a linker. In certain embodiments, the linker comprises an amino acid sequence selected from the group consisting of (GGGS)n (SEQ ID NO: 70), (GGGGS)n (SEQ ID NO: 71), (G)n (SEQ ID NO: 72), (EAAAK)n (SEQ ID NO: 73), (GGS)n (SEQ ID NO: 74), (SGGS)n (SEQ ID NO: 75), SGSETPGTSESATPES (XTEN linker) (SEQ ID NO: 76), SGSETPPKKKRKVGGSPKKKRKVGTSESATPES (2X linker) (SEQ ID NO: 77), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 78), (XP)nmotif, and any combination thereof, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. Additionally or alternatively, in some embodiments of the fusion proteins disclosed herein, the length of the linker is about 15 to about 40 amino acids.

[0016] Additionally or alternatively, in some embodiments, the general structure of the fusion proteins of the present technology is selected from the group consisting of:NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-COOH,NH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence] -COOH,-4-4856-2302-0756.1Atty. Dkt. No.: 115872-3287NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-[subcellular-localization sequence] -COOH,NH2-[subcellular-localization sequence]-[ subcellular-localization sequence]- [nuclease-defective Casl3 domain]-[PKR double-stranded RBP domain]-COOH, andNH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence]-[subcellular-localization sequence]-COOH, and

[0017] wherein each instance of comprises an optional linker, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

[0018] In some embodiments, the fusion proteins of the present technology comprise the amino acid sequence of any of SEQ ID NOs: 79-86. In certain embodiments of the fusion protein disclosed herein, the nuclease-defective Cast 3 domain is configured to specifically bind to a target RNA when the fusion protein is complexed with a guide RNA that is bound to the target RNA.

[0019] Also disclosed herein are polynucleotides comprising an open reading frame that encodes any and all embodiments of the fusion protein of the present technology. Additionally or alternatively, in some embodiments, the open reading frame is operably linked to an expression control sequence. The expression control sequence may be a tissuespecific promoter, a heterologous promoter, an inducible promoter or a constitutive promoter. In another aspect, the present disclosure provides expression vectors that comprise a polynucleotide encoding any of the fusion proteins described herein.

[0020] Also provided herein are host cells comprising a fusion protein of the present technology, a complex comprising a fusion protein of the present technology and a gRNA, a polynucleotide encoding a fusion protein of the present technology, and / or a vector that expresses such a polynucleotide. The host cells may be cancer cells, embryonic stem cells, proliferating cells, or differentiated cells.

[0021] In one aspect, the present disclosure provides kits comprising an expression vector or a host cell that includes a nucleic acid sequence encoding any of the fusion proteins described herein and instructions for use. In certain embodiments, the expression vector further comprises a nucleic acid sequence that encodes a gRNA that binds to a target RNA.-5-4856-2302-0756.1Atty. Dkt. No.: 115872-3287In other embodiments, the kit further comprises a second expression vector comprising a nucleic acid sequence that encodes a gRNA that binds to a target RNA.

[0022] In one aspect, the present disclosure provides complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to the nuclease defective Cast 3 domain of the fusion protein. The guide RNA is configured to specifically bind to and form a duplex with a target RNA. In some embodiments, the PKR double-stranded RBP domain of the fusion protein binds to the duplex between the guide RNA and the target RNA. In some embodiments, the guide RNA comprises a nucleotide sequence of any one of the guide RNA sequences described herein (e.g., SEQ ID NOs: 17-26, or 29-68).

[0023] Additionally or alternatively, in some embodiments, the target RNA is immediately adjacent to a canonical PFS protospacer sequence. In some embodiments, the target RNA is an oncogene mRNA, a tumor suppressor mRNA, a transcription factor mRNA, a RNA binding protein mRNA, a Ribosomal protein mRNA, a translation regulatory protein mRNA, a splicing factor mRNA, a metabolism mRNA, a pre-mRNA transcript, an intronic RNA, a Long noncoding RNA, a microRNA, a small RNA, a Ribosomal RNA, a telomerase RNA component, a promoter RNA, or an enhancer RNA.

[0024] In one aspect, the present disclosure provides a method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising (a) contacting cells comprising a target RNA with any and all embodiments of the complex described herein under conditions that permit binding of the complex to the target RNA, wherein the complex is configured to block binding of an RBP to a binding site within the target RNA; (b) assaying the cells of step (a) for a phenotype or biomarker expression; and (c) determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (a) with control cells that are not contacted with the complex and comprise the target RNA.

[0025] In another aspect, the present disclosure provides a method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising (a) transfecting cells comprising the target RNA with a polynucleotide encoding any and all embodiments of the fusion protein described herein; (b) transfecting the cells with a guide RNA under conditions that induce formation of a ternary complex comprising the guide-6-4856-2302-0756.1Atty. Dkt. No.: 115872-3287RNA, the target RNA and the fusion protein, wherein the ternary complex is configured to block binding of an RBP to a binding site within the target RNA; (c) assaying the cells of step (b) for a phenotype or a biomarker expression; and (d) determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (b) with control cells that are not contacted with the complex and comprise the target RNA. In some embodiments, the guide RNA is configured to specifically bind to a region within the target RNA.

[0026] In any of the preceding embodiments of the methods disclosed herein, the biomarker is a genomic biomarker, a transcriptome biomarker, a proteomic biomarker, or an epigenetic biomarker. In any and all embodiments of the methods disclosed herein, the phenotype comprises viability, growth, proliferation, differentiation, translation efficiency, RNA subcellular localization, drug susceptibility or resistance, pathogen susceptibility or resistance, signal transduction, RNA stability, genomic stability, apoptosis, senescence, or cell behavior.

[0027] Additionally or alternatively, in some embodiments of the methods described herein, the cells are isolated from a healthy subject or a subject suffering from or diagnosed with a disease. In some embodiments, the disease is cancer, an autoimmune disease, a metabolic disease or a neurodegenerative disease. Examples of cancer include, but are not limited to, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, Diffuse large B-cell lymphoma (DLBCL), lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non- Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.-7-4856-2302-0756.1Atty. Dkt. No.: 115872-3287BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGs. 1A-1F: An approach to interfere with functional units in noncoding RNA sequences. FIG. 1A: illustration of dCasl3d-dsRBD fusion protein with sgRNA and RNA targeted. FIG. IB: map of p53 binding domain (MH5 in red was experimentally defined as the minimum required domain required for p53 binding) and location of sgRNAs tiling across the region. FIGs. 1C-1D: p53 RIP (FIG. 1C) and DINO and p53-dependent gene expression (FIG. ID) in cells expressing dCasl3d-dsRBD and indicated sgRNA, demonstrating that sgRNAs targeting MH5 inhibit p53 binding and impaired p53-dependent gene expression without reducing DINO RNA abundance. FIG. IE: map of NEAT1_2 3’ domain and sgRNAs targeting the region. FIG. IF: NEAT1_2 abundance in cells expressing dCasl3d-dsRBD and indicated sgRNA. sgRNAs 4 and 5 that target U Rich #2 efficiently destabilize NEAT1_2.

[0029] FIGs. 2A-2F: Defining functional protein-RNA interactions specific to distinct subcellular regions. FIGs. 2A-2B: diagram (FIG. 2A) and fluorescence confocal microscopy (FIG. 2B) of subcellular localization of dCasl3d-dsRBD in the nucleus and cytoplasm. FIG. 2C: sgRNA screen tiling 28S a portion of ES7, revealing a 60nt region in which sgRNA binding with NoLS-dCasl3d specifically and potently impairs growth. FIG. 2D: SNV and phenotypes associated with 28S ES regions essential for growth. FIGs. 2E- 2F: screen using tiling sgRNAs over MYC mRNA. PC A shows no difference at day 0, but NLS-dCasl3d and NES-dCasl3d exert orthogonal effects by day 14, which is largely due to subcellular-specific essential protein-RNA interactions in the MYC 5’UTR and 5’SS in intron 1 of MYC.

[0030] FIGs. 3A-3E define the functional role of allele-specific RBP-RNA interactions with screening-compatible approach. FIG. 3A: Genome browser view of protein contacts within the 5’UTR of MYC mRNA as identified by ARORA in two cell lines, HepG2 and K562. * indicates the site of a single nucleotide variant in one allele of MYC in HepG2 is associated with a gain of protein occupancy. FIG. 3B: Above: Position and identity of MYC 5’UTR mutations in 2 DLBL samples from TCGA. Below: MYC protein levels in MYC 5’UTR mutant samples (red) compared to samples without MYC 5’UTR mutations. Of note, the sample with the highest MYC protein abundance with 5’UTR mutation G289A also had-8-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 the lowest MYC mRNA abundance in the entire cohort, indicating that MYC protein abundance is likely the result of post-transcriptional regulatory processes. FIG. 3C: Experimental design to block RBP-RNA interactions and their function, illustrating dCasl3d- dsRBD fusion protein with sgRNA and targeted RNA. FIG. 3D: Western blot of MYC and control Actin in HepG2 cells after expression of dCasl3d-dsRBD fusion protein with each of 9 sgRNAs tiling across the region adjacent to the SNV denoted in (FIG. 3A). 2 sequential sgRNAs impair MYC protein production remaining sgRNAs had no effect relative to sgControl indicating that dCasl3d-dsRBD fusion protein is blocking a site-specific effect that promotes MYC protein accumulation. FIG. 3E: Cell growth assay in HepG2 cells after infection with dCasl3d-dsRBD and indicated sgRNA. Only sgRNAs 7 and 8, which impaired MYC protein production, significantly decreased HepG2 cell growth.

[0031] FIG. 4 illustrates how the dCasl3-dsRNA-BP fusion protein disclosed herein to stably block RBP binding in an RNA site-specific manner.

[0032] FIG. 5 shows exemplary sgRNA sequences (SEQ ID NOs 17-68) useful in the fusion proteins of the present technology.DETAILED DESCRIPTION

[0033] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0034] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation;-9-4856-2302-0756.1Atty. Dkt. No.: 115872-3287Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning,' Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells,' Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg el al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0035] Disclosed herein are dCasl3d-double stranded RNA binding domain (dsRBD) fusion proteins that are useful for identifying the functional effects / phenotypes associated with RBP-RNA interactions (PRIs). As demonstrated by the Examples herein, these results validate that the dCasl3-dsRNA-BP fusion protein compositions of the present technology can precisely attenuate functional units encoded in discreet regions of RNA, while requiring a fraction of the time, effort, and expense of current approaches, and avoiding confounding artifacts common in other approaches, such as target-independent effects due to DNA damage or unintended distruption of DNA regulatory elements that are frequent effects of type 2 CRISPR systems. See Gilbert LA, et al. Cell. 2013;154(2):442-51; Liu SJ et al., Science. 2017;355(6320); Liu Y et al, Nature biotechnology. 2018. Epub 2018 / 11 / 06. doi: 10.1038 / nbt.4283; Horlbeck MA et al., Nature biotechnology. 2020;38(5):573-6.Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.-10-4856-2302-0756.1Atty. Dkt. No.: 115872-3287

[0037] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0038] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0039] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0040] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.-11-4856-2302-0756.1Atty. Dkt. No.: 115872-3287

[0041] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0042] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer cells” includes precancerous (e.g., benign), malignant, pre- metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc., and circulating cancers such as leukemias. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer. The term “cancer cell” refers to a cell that exhibits cancer-like properties, e.g., uncontrollable reproduction, resistance to anti- growth signals, ability to metastasize, and loss of ability to undergo-12-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 programmed cell death (e.g., apoptosis) or a cell that is derived from a cancer cell, e.g., clone of a cancer cell.

[0043] The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (z.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.” For example, the sequence “5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0044] As used herein, the term “conjugated” refers to the association of two molecules by any method known to those in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for instance, hydrogen bonds, dipolar interactions, van der Waal forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0045] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." A “control nucleic acid sample” or “reference nucleic acid sample” as used herein, refers to nucleic acid molecules from a control or reference sample. In certain embodiments, the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence. In certain embodiments, the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).-13-4856-2302-0756.1Atty. Dkt. No.: 115872-3287

[0046] As used herein, “dCas protein,” “deactivated CRISPR-associated protein,” or “dead CRISPR-associated protein" refer to a Cas protein in which only endonuclease activity is inactivated. dCas protein can form a complex with gRNA to recognize and bind to a specific gene sequence. Cas protein or gene information may be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). The fusion proteins of the present disclosure include a dCas protein as one component. A dCas protein of the present disclosure includes any gene editing protein that can recognize guide RNA and bind to target DNA / RNA, and specifically, dCas9, dCasl2a (same as dCpfl), dCasl2b, dCasl3 (dCasl3a, dCasl3b, dCasl3c, dCasl3d, etc.), dCasl4, dCasX, or any variant thereof.

[0047] The term "effective amount," as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a dCasl3d-RBP fusion protein may refer to the amount of the dCasl3d-RBP fusion protein that is sufficient to bind to a target site. In some embodiments, an effective amount of a fusion protein provided herein, may refer to the amount of the fusion protein that is sufficient to stably block RBP binding to a RNA target site specifically bound by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, may vary depending on various factors as, for example, on the desired biological response on the cell or tissue being targeted, and on the agent being used.

[0048] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0049] The term "fusion protein" as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be-14-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4. sup. th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0050] Gene” as used herein refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Although a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil, z.e., "T" is replaced with "U."

[0051] The term “gene region” can refer to a range of sequences within a gene or surrounding a gene, e.g., an intron, an exon, a promoter, a 3’ untranslated region etc.

[0052] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none;-15-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0053] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15- 100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0054] The term “hybridizable” means that two polynucleotide strands of a nucleic acid are complementary at one or more nucleotide positions, e.g., the nitrogenous bases of the two-16-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 polynucleotide strands can form two or more Crick-Watson hydrogen bonds. For example, if a polynucleotide comprises 5’ ATGC 3’, it is hybridizable to the sequence 5' GCAT 3'.Under some experimental conditions, if a polynucleotide comprises 5' GGGG 3', it can also be hybridizable to the sequences 5'CCAC 3' and 5' CCCA 3', which are not perfectly complementary.

[0055] The term "non-hybridizable" means that two polynucleotide strands of a nucleic acid are non-complementary, e.g., nitrogenous bases of the two separate polynucleotide strands do not form two or more Crick-Watson hydrogen bonds under stringent hybridization conditions.

[0056] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding a fusion protein described herein or amino acid sequence of a fusion protein described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0057] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0058] As used herein, "interaction" means a series of events in which an RNA-binding protein specifically recognizes and reacts with a target RNA to form a ribonucleoprotein, wherein the target RNA and the RNA-binding protein bind directly or indirectly.-17-4856-2302-0756.1Atty. Dkt. No.: 115872-3287

[0059] The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cast 3d domain and a dsRNA RBP domain. In some embodiments, a linker joins a nuclease-defective Cast 3d domain and a dsRNA RBP domain. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45- 50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0060] As used herein, “oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. One or more bases of the oligonucleotide may also be modified to include a phosphorothioate bond (e.g., one of the two oxygen atoms in the phosphate backbone which is not involved in the intemucleotide bridge, is replaced by a sulfur atom) to increase resistance to nuclease degradation.Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 55 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide-18-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.

[0061] As used herein, the term “peptide” means a short polymer of 2-50 amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. In some embodiments, the peptide is no more than 10 amino acids in length. Peptides may contain amino acids other than the 20 gene-encoded amino acids. Peptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0062] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0063] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0064] As used herein, "RNA-binding protein" or “RBP” of the present disclosure refers to a protein involved in the formation of a ribonucleoprotein (RNP) complex by binding to single-stranded non- structural RNA or double-stranded structural RNA in a cell (J. Appl. Biol. Chem 58(3), 2015, 201-208). The RBP includes a wild-type RBP capable of interacting-19-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 with the target RNA, derivatives, variants, and domains responsible for binding functions or part of a polypeptide.

[0065] The term “stringent hybridization conditions” as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaffcPCh, pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42° C. overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.

[0066] As used herein, the term “target RNA” refers to an RNA of interest that is capable of binding to an RNA-binding protein. Target RNA may be tRNA (transfer RNA), mRNA (messenger RNA), rRNA (ribosomal RNA), various small noncoding RNAs, long noncoding RNAs, virus / bacteria, etc. dCas!3 Proteins

[0067] Exemplary nuclease defective Cast 3 amino acid sequences useful in the fusion proteins described herein are provided below:

[0068] dRfxCasl3d Casl3d amino acid sequence:

[0069] IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNE GEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKR YFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTY DEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN YGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRIT NEL TN SF SKNS A AN VN YI AETLGINP AEF AEQ YFRF SIMKEQKNLGFNITKLREVMLD RKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEK DIFVINLRGSFNDDQI<DALYYDEANRIWRI<LENIMHNII<EFRGNI<TREYI<I<I<DAPRL PRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAK FVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKAL ADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVK FVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYK-20-4856-2302-0756.1Atty. Dkt. No.: 115872-3287EKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANP KLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCT LFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFD AVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 1)

[0070] dAdmCas!3d amino acid sequence:

[0071] KRKTKAKAAGLKSVFFDQKQAVLTTFAKGNNSQIEKKVVNSEVKDLRQP PAFDLELKEKTFYISGKNNINTSRENPLASASLPLSKRQRIRAERIKRAREENRPYHNV KRVGEDDLRAKADLEKHYFGKEYSDNLKIQIIYNILDINKIISPYINDIVYSMNNLARN DEYIDGKID VIGSLS STTD YS SFMSPNKDLEKEKKF SFHRENYKKF VEASKPYMRYY GKVFIRDVKKSKLSTGKGEKIEVMYRSDEEIFTIFQILSYVAQSIMANDIGNKSSILAIE KYPARFVGFLSDLLKTKTNDVNRMFIDNNSQTNFWVLFSIFGLQDHTSGADKICRNF YDFVIKADSKNLGFSLKKIRELMLDLPNANMLRDHQFDTVRSKFYTLLDFIIYQHYLE EKSRIDNMVEKLRMTLKEEEKEVLYAAEAKIVWNAIGAKVINKLVPMMNGDALKEI KRKNRDRKLPQSVIATVQVNSDANVFSGLIYFLTLFLDGKEINEMVSNLITKFENIDSL LHVDREIYKSDEKDLDLEIEKLALFFKGVVRPNAKTDTGAGEISKSFSIFQSAERIIEEL KFIKNVTRMDNEIFPSEGVFLDAANVLGVRGDDFDFSNEFVGDDLHSDANKKIINKIN GTKEDRNLRNFIINNVVKSRRFQYIARHMNTHYVKQLANNETLNRFVLNKMGDAKII NRYYESISGNTPNIEVRSQIDYLVKRLRSFSFEDLNDVKQKVRPGTNESIEKEKKKAL VGLCLTIQYLVYKNLVNINARYTTAFYCLERDSKLKGFGVDVWRDFESYTALTNHFI KEGYLPVRKAEILRANLKHLDCEDGFKYYANQVTALNAIRVAYKYINEIKSVHSYFALYHYIMQRHLYDSLQAKAKDSSGFVIDALKKSFEHKIYSKDLLHVLHSPFGYNTARY KNLSIEALFDKN (SEQ ID NO: 2)

[0072] dLwCas!3a amino acid sequence:

[0073] KVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNAS EEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNK NSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEK VGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKI REYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEE LNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNK-21-4856-2302-0756.1Atty. Dkt. No.: 115872-3287LDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETEN ENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDN KNEIEDFFANIDEAISSIAHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKL KIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFF WSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHY KYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLK YIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKI LKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNR VTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGM LNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDE DYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLK GEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKL KQEKKDLYIANYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYG FVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE(SEQ ID NO: 3)

[0074] dCasl3b amino acid sequence:

[0075] MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNR VEVNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINN YYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNG DTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPK DRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDR FVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRL EEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMF INDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHN RYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDM LTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGE NKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFAR SIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIY SEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFY QWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIR-22-4856-2302-0756.1Atty. Dkt. No.: 115872-3287SNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELAD FDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASD I<RIGNLLELVGSDIVSI<EDIMEEFNI<YDQCRPEISSIVFNLEI<WAFDTYPELSARVDRE (SEQ ID NO: 87)

[0076] In some embodiments, the dCasl3 domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the dCasl3 domain of any one of SEQ ID NOs: 1-3 or 87. In some embodiments, the dCasl3 domain comprises the amino acid sequence of any one of SEQ ID NOs: 1-3 or 87.Subcellular Localization Sequences

[0077] Exemplary subcellular localization amino acid sequences useful in the fusion proteins described herein are provided below:-23-4856-2302-0756.1Atty. Dkt. No.: 115872-3287Fusion Proteins of the Present Technology

[0078] In one aspect, the present disclosure provides a fusion protein comprising a double-stranded RNA binding protein (RBP) domain, a nuclease-defective Cast 3 domain, and one or more subcellular-localization sequences. In some embodiments, the doublestranded RBP domain is a PKR double-stranded RBP domain. In further embodiments, the PKR double-stranded RBP domain comprises the amino acid sequence ofAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGR SKKEAKNAAAKLAVEILNKEKKA (SEQ ID NO: 69).

[0079] In other embodiments, the double-stranded RBP domain is the double stranded RNA binding motif of a mammalian protein, such as: ADAR, RNase III, or DICER. Exemplary amino acid sequences of the double stranded RNA binding motif of these mammalian proteins are provided below:

[0080] ADAR:

[0081] PISGLLEYAQFASQTCEFNMIEQSGPPHEPRFKFQVVINGREFPPAEAGSKK VAKQDAAMKAMTILL (SEQ ID NO: 88)

[0082] RNase III:

[0083] PKSQLQQCCLTLRTEGKEPDIPLYKTLQTVGPSHARTYTVAVYFKGERIGC GKGPSIQQAEMGAAMDALEKYN (SEQ ID NO: 89)

[0084] DICER:

[0085] PRSP VRELLEMEPET AKF SP AERT YDGKVRVT VE VVGKGKFKGVGRS YRIAKSAAARRALRSLK (SEQ ID NO: 90)

[0086] Additionally or alternatively, in some embodiments of the fusion protein disclosed herein, the nuclease-defective Casl3 domain is dCasl3a, dCasl3b, or dCasl3d. In certain embodiments, the nuclease-defective Casl3 domain comprises the amino acid sequence of any of SEQ ID NOs: 1-3 or 87. In some embodiments, the dCasl3 domain is at least 80%, at-24-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the dCasl3 domain of any one of SEQ ID NOs: 1-3 or 87.

[0087] The PKR double-stranded RBP domain may be fused to the N-terminus or the C- terminus of the nuclease-defective Cast 3 domain. In any of the preceding embodiments of the fusion proteins described herein, the nuclease-defective Cast 3 domain and the PKR double-stranded RBP domain are fused via a linker, while in other embodiments the nuclease-defective Cast 3 domain and the PKR double-stranded RBP domain are fused directly to one another. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of (GGGS)n (SEQ ID NO: 70), (GGGGS)n (SEQ ID NO: 71), (G)n (SEQ ID NO: 72), (EAAAK)n (SEQ ID NO: 73), (GGS)n (SEQ ID NO: 74), (SGGS)n (SEQ ID NO: 75), SGSETPGTSESATPES (XTEN linker) (SEQ ID NO: 76), SGSETPPKKKRKVGGSPKKKRKVGTSESATPES (2X linker) (SEQ ID NO: 77), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 78), (XP)nmotif, and any combination thereof, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. Additionally or alternatively, in some embodiments of the fusion proteins disclosed herein, the length of the linker is about 15 to about 40 amino acids.

[0088] Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, the entire contents of which are incorporated herein by reference. Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure.

[0089] The one or more subcellular-localization sequences may be fused to the N- terminus or the C-terminus of the fusion protein. In some embodiments, the one or more subcellular-localization sequences may be fused to the N-terminus or the C-terminus of the PKR double-stranded RBP domain. Additionally or alternatively, in some embodiments, the one or more subcellular-localization sequences may be fused to the N-terminus or the C- -25-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 terminus of the nuclease-defective Cast 3 domain. In some embodiments, the one or more subcellular-localization sequences are fused to any of the PKR double-stranded RBP domain, or the nuclease-defective Casl3 domain via one or more linkers. In other embodiments, the subcellular-localization sequences are fused to any of the PKR double-stranded RBP domain, or the nuclease-defective Cast 3 domain without a linker.

[0090] In any of the preceding embodiments of the fusion protein disclosed herein, the one or more subcellular-localization sequences are selected from the group consisting of nuclear localization sequences, Endoplasmic Reticulum membrane localization sequences, mitochondrial localization sequences, cytoplasmic localization sequences, nucleolus localization sequences, and plasma membrane localization sequences. Additionally or alternatively, in some embodiments, the one or more subcellular-localization sequences comprise the amino acid sequences of any of SEQ ID NOs: 4-16.

[0091] Additionally or alternatively, in certain embodiments, the one or more subcellular- localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain. In any of the above embodiments of the fusion proteins disclosed herein, the one or more subcellular-localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain and the C-terminus of the PKR double-stranded RBP domain. In other embodiments of the fusion proteins disclosed herein, one or more subcellular-localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain and the N- terminus of the PKR double-stranded RBP domain.

[0092] Additionally or alternatively, in some embodiments, the one or more subcellular- localization sequences are located at the N-terminus of the nuclease-defective Casl3 domain. In any of the above embodiments of the fusion proteins disclosed herein, the one or more subcellular-localization sequences are located at the N-terminus of the nuclease-defective Cast 3 domain and the N-terminus of the PKR double-stranded RBP domain. In other embodiments of the fusion proteins disclosed herein, the one or more subcellular-localization sequences are located at the N-terminus of the nuclease-defective Cast 3 domain and the C- terminus of the PKR double-stranded RBP domain.

[0093] In certain embodiments, the fusion protein of the present technology comprises one subcellular-localization sequence located at the N-terminus of the nuclease-defective-26-4856-2302-0756.1Atty. Dkt. No.: 115872-3287Casl3 domain and / or the N-terminus of the PKR double-stranded RBP domain. In other embodiments, the fusion protein of the present technology comprises one subcellular- localization sequence located at the C-terminus of the nuclease-defective Cast 3 domain and / or the C-terminus of the PKR double-stranded RBP domain.

[0094] Additionally or alternatively, in certain embodiments, the fusion protein of the present technology comprises at least two subcellular-localization sequences. The at least two subcellular-localization sequences may be identical or different. In some embodiments, the at least two subcellular-localization sequences are located at the N-terminus of the nuclease-defective Casl3 domain and / or the N-terminus of the PKR double-stranded RBP domain. In certain embodiments, the at least two subcellular-localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain and / or the C-terminus of the PKR double-stranded RBP domain.

[0095] In other embodiments, one of the at least two subcellular-localization sequences is located at the N-terminus of the nuclease-defective Cast 3 domain and the N-terminus of the PKR double-stranded RBP domain, and one of the at least two subcellular-localization sequences is located at the C-terminus of the nuclease-defective Cast 3 domain and the C- terminus of the PKR double-stranded RBP domain.

[0096] Other exemplary features that may be present in the fusion protein described herein, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc- tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S- transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more suitable protein tags.

[0097] In any of the preceding embodiments, the fusion proteins of the present technology further comprise a detectable protein, such as a bioluminescent protein, a-27-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 fluorescent protein, a chemiluminescent protein, or any combination thereof. Examples of bioluminescent protein include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent protein include, but are not limited to, P-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent protein include, but are not limited to, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, HIKOK, mK02, mOrange, m0range2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKatel, LSS-mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS- CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0098] Additionally or alternatively, in some embodiments, the PKR double-stranded RBP domain and / or the nuclease-defective Casl3 domain and the detectable protein are linked via a linker. In certain embodiments, the linker comprises an amino acid sequence selected from the group consisting of (GGGS)n (SEQ ID NO: 70), (GGGGS)n (SEQ ID NO: 71), (G)n (SEQ ID NO: 72), (EAAAK)n (SEQ ID NO: 73), (GGS)n (SEQ ID NO: 74), (SGGS)n (SEQ ID NO: 75), SGSETPGTSESATPES (XTEN linker) (SEQ ID NO: 76), SGSETPPKKKRKVGGSPKKKRKVGTSESATPES (2X linker) (SEQ ID NO: 77), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 78), (XP)nmotif, and any combination thereof, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. Additionally or alternatively, in some embodiments of the fusion proteins disclosed herein, the length of the linker is about 15 to about 40 amino acids.

[0099] Additionally or alternatively, in some embodiments, the general structure of the fusion proteins of the present technology is selected from the group consisting of:4856-2302-0756.1Atty. Dkt. No.: 115872-3287NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-COOH,NH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence] -COOH,NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-[subcellular-localization sequence] -COOH,NH2-[subcellular-localization sequence]-[ subcellular-localization sequence]- [nuclease-defective Casl3 domain]-[PKR double-stranded RBP domain]-COOH, andNH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence]-[subcellular-localization sequence]-COOH, and wherein each instance of comprises an optional linker, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

[0100] It should be appreciated that any of the proteins provided in any of the general architectures of exemplary fusion proteins may be connected by one or more of the linkers provided herein. In some embodiments, the linkers are the same. In some embodiments, the linkers are different. In some embodiments, one or more of the proteins provided in any of the general architectures of exemplary fusion proteins are not fused via a linker.

[0101] Exemplary amino acid sequences of the fusion proteins of the present technology include SEQ ID NOs: 79-86.

[0102] dCasl3d-dsRBD (generic localization signal scheme 1 : N- & C-terminal tags) (SEQ ID NO: 79):M [Affinity_Tag ] GSKRTADGSEFES [ localization_tag] EAS IEKKKSFAKGMGVKSTLVS GSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAWANNPLY TGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIG FGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINY GNECYDILALLSGLAHWVVANNEEESRISRTWL YNLDKNLDNEYIS TLNYL YDRITNEL TNSFSKNSA ANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLEN IMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQ SFLKVMPL IGVNAKFVEE YAFFKDSAKIADELRL IKSFARMGEPIADARRAMYIDAIRILGTNL S YDE-29-4856-2302-0756.1Atty. Dkt. No.: 115872-3287LKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAWKFVLGRIA DIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREK FKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGID ETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNT KWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSS GKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNSGSGAG DLSAGFFMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLA VEILNKEKKAGSKRTADGSEFES [ localization tag ] GSGATNFSLLKQAGDVEENPGPEFMVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0103] dCasl3d-dsRBD (generic localization signal scheme 2: two N-terminal tags) (SEQ ID NO: 80):M [Affinity Tag ] GSKRTADGSEFES [ localization tag] GSGS [ localization tag ] GSGEAS IEKKKS FAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS IRSVNEGEAFSAEMADKN AGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEK I LAE YI TNAA YAVNNISGLDKDIIGFGKFS TVYTYDEFKDPEHHRAAFNNNDKL INAIKAQYDEFDNF LDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWWANNEEESRISRTWLYNLDKNLDNE YISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREV MLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLR GSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYA L TMFLDGKEINDLL TTL INKFDNIQS FLKVMPL IGVNAKFVEE YAFFKDSAKIADELRL IKS FARMGE PIADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIR YGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITG MNYDQFDKKRSVIEDTGRENAEREKFKKIISL YL TVIYHILKNIVNINARYVIGFHCVERDAQL YKEK GYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEK KAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAE VNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEAL FA AAAKFAKAKKKVSG SGSGAGDLSAGFFMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVI IDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAGSKRTADGSEFESATNFSLLKQAGDVEENPGPE FMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDG-30-4856-2302-0756.1Atty. Dkt. No.: 115872-3287NILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0104] dCasl3d-dsRBD (generic localization signal scheme 3: two C-terminal tags) (SEQ ID NO: 81):M [ Af f inity_Tag ] GSKRTADGSEFESEAS IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLE KIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAWANNPLYTGPVQQDMLGLKETLEKR YFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEH HRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWV VANNEEESRISRTWL YNLDKNLDNEYIS TLNYL YDRITNEL TNSFSKNSAANVNYIAETLGINPAEFA EQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKV AAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKK KDAPRL PRIL PAGRDVSAFSKLMYAL TMFLDGKEINDLLTTL INKFDNIQSFLKVMPL IGVNAKFVEE YAFFKDSAKIADELRL IKSFARMGEPIADARRAMYIDAIRILGTNL S YDELKALAD TFSLDENGNKLK KGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAWKFVLGRIADIQKKQGQNGKNQIDRYY ETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKN IVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERA KESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTC TLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYND RLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNSGSGAGD SAGFFMEEIJSTYRQKQ GWLKYQELPNSGPPHDRRFTFQVI IDGREFPEGEGRSKKEAKNAAAKLAVE ILNKEKKAGS KRTADG SEFES [ localization_tag ] GSGS [ localization tag ] GSGATNFSLLKQAGDVEENPGPE FMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY GVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDG NILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0105] dCasl3d-dsRBD (generic localization signal scheme 3: N-terminal tags) (SEQ ID NO: 82):[ localization_tag] GSGS [Af f inity_Tag] GSKRTADGSEFESEAS IEKKKSFAKGMGVKST LVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANN PL YTGPVQQDMLGLKE TLEKRYFGESADGNDNICIQVIHNILDIEKILAE YI TNAA YAVNNISGLDKD IIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYI INYGNECYDILALLSGLAHWWANNEEESRISRTWL YNLDKNLDNEYIS TLNYL YDRITNEL TNSFSK NSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRT-31-4856-2302-0756.1Atty. Dkt. No.: 115872-3287KVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRK LENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFD NIQS FLKVMPL IGVNAKFVEE YAFFKDSAKIADELRL IKS FARMGEPIADARRAMYIDAIRILGTNL S YDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLG RIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAE REKFKKIISL YL TVIYHILKNIVNINARYVIGFHCVERDAQL YKEKGYDINLKKLEEKGFSSVTKLCA GIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYL RNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYE KS SGKVSE YFDAVNDEKKYNDRLLKL LCVPFG YCIPRFKNL S IE AL FDRNEAAKFDKEKKKVSGNSG SGAGDLSAGFFMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAA KLAVE I LNKE KKAG SKRTADGSEFESGSGSGS GATNFSLLKQAGDVEENPGP E FMVSKGEELFTGVVP ILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQH DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH MVLLEFVTAAGITLGMDELYK*

[0106] dCasl3d-dsRBD (generic localization signal scheme 3: C-terminal tags) (SEQ ID NO: 83):M [ Af f inity_Tag ] GSKRTADGSEFESEAS IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLE KIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAWANNPLYTGPVQQDMLGLKETLEKR YFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEH HRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWV VANNEEESRISRTWL YNLDKNLDNEYIS TLNYL YDRITNEL TNSFSKNSAANVNYIAETLGINPAEFA EQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKV AAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKK KDAPRL PRIL PAGRDVSAFSKLMYAL TMFLDGKEINDLLTTL INKF DN IQS FLKVMPL IGVNAKFVEE YAFFKDSAKIADELRL IKS FARMGEPIADARRAMYIDAIRILGTNL S YDELKALAD TFSLDENGNKLK KGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAWKFVLGRIADIQKKQGQNGKNQIDRYY ETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKN IVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERA KESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTC TLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYND RLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNSGSGAGD SAGFFMEEIJSTYRQKQ GWLKYQELPNSGPPHDRRFTFQVI IDGREFPEGEGRSKKEAKNAAAKLAVE ILNKEKKAGS KRTADG-32-4856-2302-0756.1Atty. Dkt. No.: 115872-3287SE FE SGSGSGSGATNFSLLKQAGDVEENPGPE FMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK GSGS [ localization tag] *

[0107] NLS - dCasl3d-dsRBD (nuclear localization signal) (SEQ ID NO: 84):MGKPIPNPLLGLDSTGSKRTADGSEFESPKKJCRKyEAS IEKKKSFAKGMGVKSTLVSGSKVYMTTFAE GSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGL KE TLEKRYFGESADGNDNICIQVIHNILDIEKILAE YI TNAA YAVNNISGLDKDIIGFGKFS TVYTYD EFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALL SGLAHWWANNEEESRISRTWL YNLDKNLDNEYISTLNYL YDRITNEL TNSFSKNSAANVNYIAETLG INPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYY IEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGN KTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGV NAKFVEE YAFFKDSAKIADELRL IKS FARMGEPIADARRAMYIDAIRILGTNL S YDELKALAD TFSLD ENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGK NQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTV IYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVE KEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLL RIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVN DEKKYNDRLLKL LCVPFG YCIPRFKNL S IE AL FDRNEAAKFDKEKKKVSGNSG S GAGDLS GFFMEEL NTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAG SKRTADGSEFESPKKKRKVGSGATNFSLLKQAGDVEENPGPEFMVSKGEELFTGVVPILVELDGDVNG HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGY VQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG ITLGMDELYK*

[0108] NES - dCasl3d-dsRBD (nuclear export signal, for cytoplasmic localization) (SEQ ID NO: 85):MGKPIPNPLLGLDSTGSKRTADGSEFESLPPL_ERLTLEAS IEKKKS FAKGMGVKSTLVSGSKVYMTTF AEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDML-33-4856-2302-0756.1Atty. Dkt. No.: 115872-3287GLKE TLEKRYFGESADGNDNICIQVIHNILDIEKILAE YI TNAA YAVNNISGLDKDIIGFGKFS TVYT YDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILA LLSGLAHWWANNEEESRISRTWL YNLDKNLDNEYISTLNYL YDRITNEL TNSFSKNSAANVNYIAET LGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYR YYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFR GNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLI GVNAKFVEE YAFFKDSAKIADELRL IKS FARMGEPIADARRAMYIDAIRILGTNL S YDELKALAD TFS LDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQN GKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYL TVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKD VEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIRED LLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDA VNDEKKYNDRLLKL LCVPFG YCIPRFKNL S IE AL FDRNEAAKFDKEKKKVSGNSG S GAGDLS AGFFME ELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKK AGSKRTADGSEFESLPPLERLTLGSGATNFSLLKQAGDVEENPGPEFMVSKGEELFTGVVPILVELDG DVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVOCFSRYPDHMKQHDFFKSAM PEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADK QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV TAAGITLGMDELYK*

[0109] NoLS - dCasl3d-dsRBD (nucleolar localization domain 3x NIK) (SEQ ID NO: 86):AS IEKKKS FAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS IRSVNEGEAFSAEMADKNAGYK IGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAE YI TNAA YAVNNISGLDKDIIGFGKFS TVYTYDEFKDPEHHRAAFNNNDKL INAIKAQYDEFDNFLDNP RLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWWANNEEESRISRTWLYNLDKNLDNEYIST LNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDR KDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFN DDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMF LDGKEINDLL TTL INKFDNIQS FLKVMPL I GVNAKFVEE YAFFKDSAKIADELRL IKS FARMGEPIAD ARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDP AHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYD QFDKKRSVIEDTGRENAEREKFKKIISL YL TVIYHILKNIVNINARYVIGFHCVERDAQL YKEKGYDI NLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEE-34-4856-2302-0756.1Atty. Dkt. No.: 115872-3287FTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSY FQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRN EAAKFDKEKKKVSGNSGSGAGDLSAGFFMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGR EFPEGEGRSKKEAKNAAAKIAVEILJSrKEKKAGSKRTADGSEFESRMRJCKRKKKLRnAtMRKKRKKJCLR J LJ^RKKRKKKL_R I LG S GATNFSLLKQAGDVEENPGPE FMVSKGEELFTGVVPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER TI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVOLADHYOONTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG MDELYK*

[0110] For SEQ ID NOs: 79-86, dCasl3d - italics; PKR dsRNA binding domain - boldface, Spacer-single underline, Fluorescent Protein- double underline, affinity tag - boldface dotted underline, localization sequences - dash underline.Fusion Protein Complexes with Guide RNAs

[0111] In one aspect, the present disclosure provides complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to the nuclease defective Cast 3 domain of the fusion protein. The guide RNA is configured to specifically bind to and form a duplex with a target RNA. In some embodiments, the PKR double-stranded RBP domain of the fusion protein binds to the duplex between the guide RNA and the target RNA.

[0112] In some embodiments, the guide RNA is about 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target RNA. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target RNA.

[0113] Additionally or alternatively, in some embodiments, the target RNA is immediately adjacent to a canonical PFS protospacer sequence. In some embodiments, the target RNA is an oncogene mRNA, a tumor suppressor mRNA, a transcription factor mRNA, a RNA binding protein mRNA, a Ribosomal protein mRNA, a translation regulatory protein mRNA, a splicing factor mRNA, a metabolism mRNA, a pre-mRNA transcript, an intronic-35-4856-2302-0756.1Atty. Dkt. No.: 115872-3287RNA, a Long noncoding RNA, a microRNA, a small RNA, a Ribosomal RNA, a telomerase RNA component, a promoter RNA, or an enhancer RNA.

[0114] In any and all embodiments of the complexes disclosed herein, the guide RNA is complementary to a target RNA associated with a disease or disorder (e.g., cancer). In some embodiments, the guide RNA is complementary to a target RNA comprising a mutation that is associated with a disease or disorder (e.g., cancer). In some embodiments, the guide RNA comprises a nucleotide sequence of any one of the guide RNA sequences described herein (e.g., SEQ ID NOs: 17-26, or 29-68).Methods of the Present Technology

[0115] In one aspect, the present disclosure provides a method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising (a) contacting cells comprising a target RNA with any and all embodiments of the complex described herein under conditions that permit binding of the complex to the target RNA, wherein the complex is configured to block binding of an RBP to a binding site within the target RNA; (b) assaying the cells of step (a) for a phenotype or biomarker expression; and (c) determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (a) with control cells that are not contacted with the complex and comprise the target RNA.

[0116] In another aspect, the present disclosure provides a method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising(a) transfecting cells comprising the target RNA with a polynucleotide encoding any and all embodiments of the fusion protein described herein; (b) transfecting the cells with a guide RNA under conditions that induce formation of a ternary complex comprising the guide RNA, the target RNA and the fusion protein, wherein the ternary complex is configured to block binding of an RBP to a binding site within the target RNA; (c) assaying the cells of step(b) for a phenotype or a biomarker expression; and (d) determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (b) with control cells that are not contacted with the complex and comprise the target RNA. In some embodiments, the guide RNA is configured to specifically bind to a region within the target RNA.-36-4856-2302-0756.1Atty. Dkt. No.: 115872-3287

[0117] In any of the preceding embodiments of the methods disclosed herein, the biomarker is a genomic biomarker, a transcriptome biomarker, a proteomic biomarker, or an epigenetic biomarker. In any and all embodiments of the methods disclosed herein, the phenotype comprises viability, growth, proliferation, differentiation, translation efficiency, RNA subcellular localization, drug susceptibility or resistance, pathogen susceptibility or resistance, signal transduction, RNA stability, genomic stability, apoptosis, senescence, or cell behavior.

[0118] Additionally or alternatively, in some embodiments of the methods described herein, the cells are isolated from a healthy subject or a subject suffering from or diagnosed with a disease. In some embodiments, the disease is cancer, an autoimmune disease, a metabolic disease or a neurodegenerative disease. Examples of cancer include, but are not limited to, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, Diffuse large B-cell lymphoma (DLBCL), lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non- Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.Kits, Vectors, and Host Cells

[0119] Also disclosed herein are polynucleotides comprising an open reading frame that encodes any and all embodiments of the fusion protein of the present technology. Additionally or alternatively, in some embodiments, the open reading frame is operably linked to an expression control sequence. The expression control sequence may be a tissuespecific promoter, a heterologous promoter, an inducible promoter or a constitutive promoter. In another aspect, the present disclosure provides expression vectors that comprise a polynucleotide encoding any of the fusion proteins described herein.

[0120] Also provided herein are host cells comprising a fusion protein of the present technology, a complex comprising a fusion protein of the present technology and a gRNA, a-37-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 polynucleotide encoding a fusion protein of the present technology, and / or a vector that expresses such a polynucleotide. The host cells may be cancer cells, embryonic stem cells, proliferating cells, or differentiated cells.

[0121] In one aspect, the present disclosure provides kits comprising an expression vector or a host cell that includes a nucleic acid sequence encoding any of the fusion proteins described herein and instructions for use. In certain embodiments, the expression vector further comprises a nucleic acid sequence that encodes a gRNA that binds to a target RNA. In other embodiments, the kit further comprises a second expression vector comprising a nucleic acid sequence that encodes a gRNA that binds to a target RNA.

[0122] Additionally or alternatively, in some embodiments, the kits may comprise an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of an RNA sequence identical or complementary to a target RNA into the guide RNA backbone.

[0123] In another aspect, the present disclosure provide kits that include one or more of the sgRNAs described herein (e.g., any one or more of SEQ ID NOs: 17-26, or 29-68).EXAMPLES

[0124] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: Materials and Methods

[0125] Cell lines. HepG2, K562, 293T, were obtained from ATCC and grown in recommended base media with 10% FBS.

[0126] Lentiviral preparation. Lentiviral plasmids expressing (1) dCasl3d-dsRBD or (2) sgRNAs were transfected into 293T cells with packaging plasmids by the Calcium Phosphate method. 24 and 48 hours after transfection, virus-containing medium was harvested.

[0127] Lentiviral infections and selection. HepG2 or 293 T cell lines were infected first with dCasl3d-dsRBD-P2A-GFP and selected with antibiotic puromycin for 3 days, sorted with FACS to obtain a consistent population highly expressing dCasl3d-dsRBD and GFP, then maintained in puromycin medium. sgRNA expressing lentiviral libraries expressing BFP were infected in cell lines expressing dCasl3d-dsRBD, selected for 3 days with-38-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 hygromycin, and then sorted on day 5 post-infection by FACS to obtain consistent population of GFP+, BFP+ cells. 3 million sorted cells were saved as TO. 3-5 million cells were maintained in culture with puromycin and hygromycin selection for 7 days (T7) and 14 days (T14).

[0128] sgRNA library preparation, sequencing, and analyses. Genomic DNA was extracted from infected cells at TO, T7, and T14 using Qiagen Genomic DNA kit. sgRNA sequences were amplified using primers targeting the lentiviral vector adjacent to the sgRNA variable spacer. Each library was sequenced to approximately 20 million unique reads. sgRNA sequencing was analyzed by MAGeCK to identify regions of sequential sgRNAs that elicited growth inhibitory effects.

[0129] Western blot. Cells were lysed in SDS running buffer and protein extracts run on a 12% PAGE gel. Antibodies for MYC and ACTIN were used to quantify protein and LICOR secondary antibodies were used to image and quantify protein levels on a LICOR Odyssey instrument.

[0130] Immunofluorescence and Microscopy . 293T cells were infected with lentiviral expression systems with NLS- dCasl3d-dsRBD and NES- dCasl3d-dsRBD for 3 days, then fixed with paraformaldehyde. Cells were lysed and an antibody against V5 was used for immunofluorescence of the dCasl3d-dsRBD fusion protein. Fluorescence images were acquired with an inverted Zeiss LSM880 (Carl Zeiss Microscopy GmbH) equipped with an Airy Scan detector (gain 850, digital gain 1) in super resolution mode.

[0131] RNA isolation and qRT-PCR. RNA was isolated from cells using the Qiagen RNeasy Plus kit. Indicated transcripts were quantified by qRT-PCR using SYBR Green RT- PCR Master Mix (ThermoFisher) on a QuantStuido 6 instrument.

[0132] p53 RNA immunoprecipitation. Cells were transfected with dCasl3d-dsRBD and sgRNAs targeting DINO. Three days later, cells were crosslinked with paraformaldehyde for 10 minutes. RNA immunoprecipitation was subsequently performed using the ActiveMotif RNA ChlP-IT kit, and anti-p53 antibody DO-1.-39-4856-2302-0756.1Atty. Dkt. No.: 115872-3287Example 2: dCas!3 Assay to Examine the Functional Effects of Interfering with Individual PRIs

[0133] Characterizing the functional role of functional units composed of ncRNA and interacting proteins provides the opportunity to gain novel insights into the molecular pathogenesis of cancer. We have developed an RNA targeting CRISPR that precisely attenuates functional units in endogenous ncRNAs that does not alter genetic sequence or transcript abundance, thus avoiding the prevalent artifacts generated by other approaches used to interrogate the function of ncRNA sequences. This approach employs a nuclease deficient variant of Cast 3d (dCasl3d) fused to a double stranded RNA binding domain (dsRBD) of PKR that stabilizes the site-specific interaction dsRNA generated by the sgRNA annealing with the target ncRNA (FIG. 1A). This obstructs native interactions between ncRNA and proteins and can interfere with secondary structure to probe for function. Using this technology, we have validated that directing dCasl3d-dsRBD to the p53 binding site on DINO specifically obstructs DINO’s binding to p53 and inhibits DINO’s downstream regulation of p53 signaling without altering the abundance of DINO in cells (FIGs. 1B-1D). Similarly, we obtained a high-resolution annotation of the structural element critical for the stability of the long noncoding RNA NEAT1 in vivo. This approach revealed that a lOnt U- rich motif #2 (U2) was the critical element for stabilization of the transcript (FIGs. 1E-1F). These results validate that this approach can precisely attenuate functional units encoded in discreet regions of a ncRNA, replicating prior observations, but requiring a fraction of the time, effort, and expense of current approaches, and avoiding confounding artifacts common in other approaches.

[0134] Diverse RNA species interact with distinct sets of RNA binding proteins depending on their specific subcellular localization. For example, in the process of generating mRNA, newly transcribed pre-mRNA associates with a variety of nuclear regulatory proteins to ensure RNA processing including 5’ capping, 3’ polyadenylation, and splicing to generate mature transcripts while protein-RNA interactions in the cytosol may alternatively regulate mRNA stability, localization, and translation efficiency. Dissecting the subcellular location in which specific protein-RNA interaction are required is not possible with current approaches. We reasoned that restricting the localization of dCasl3d-dsRBD to specific subcellular regions could identify distinct, sub-cellular region-specific protein-RNA interactions required for the function of an RNA. We generated dCasl3d-dsRBD constructs-40-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 tagged with three distinct subcellular localization tags to direct the fusion protein to a specific subcellular region and validated appropriate subcellular localization by immunofluorescence (FIGs. 2A-2B) We applied this experimental approach for annotation of essential functional regions in the poorly characterized expansion segments of the large ribosomal RNA subunit. We expressed a library of sgRNAs tiling across these expansion segments at 5 nucleotide resolution along with either cytoplasm localized NES-dCasl3d-dsRBD or nucleolus-localized NoLS-dCasl3d-dsRBD and measured cellular growth. When expressed with NoLS- dCasl3d-dsRBD localized to the nucleolus, the site of ribosome biogenesis, multiple independent sgRNAs targeting three discreet regions of the expansion segments substantially impaired cell growth while the vast majority of sgRNAs had no effect on cell growth (FIG. 2C). These results of FIG. 2D are corroborated in Rothschild et al., bioRxiv (2023), which has shown that these three discreet sites, each less than 60 nucleotide length, account for nearly 1 / 3 of the recurrent, expressed rRNA sequence variants, and affect key essential sequences in rRNA expansion segments. In contrast, expression of the sgRNA library with NES-dCasl3d resulted in no growth impairment at any site. This demonstrates that perturbing these specific sequences in the nucleolus is critical for the ability to identify their functionality in cells.

[0135] Next, we examined the ability of this system to identify functionally relevant protein-RNA interactions that are distinct between two different subcellular domains. We focused on the MYC mRNA, a universally essential gene. We expressed a library of sgRNAs tiling across the full length of the MYC transcript at 5 nucleotide resolution. Expression of the sgRNA library with NES-dCasl3d-dsRBD identified a set of discreet RBP bound sites in the 5’UTR and first intron of MYC mRNA that are necessary for cell growth (FIGs. 2E-2F), but in a sub-cellular region specific manner. Expressing this sgRNA library along with NLS- dCasl3d-dsRBD identified a distinct region that corresponds to the well-defined 5’ Splice Site (5’ SS) in the first intron of MYC are required for cell growth. Consistent with the exclusive role for this intronic sequence in nucleus, sgRNAs targeting the 5’SS had no impact on the growth of cells when expressed with cytosol-localized fusion protein (NES-daCasl3d- dsRBD). In contrast, specific sets of sgRNAs targeting the MYC 5’UTR IRES element, which enhances MYC translation which occurs exclusively in the cytoplasm, impaired cell growth when expressed with NES-daCasl3d-dsRBD, but not when expressed with NLS--41-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 daCasl3d-dsRBD. This functional sites in the 5’UTR IRES correspond to protein-bound sites identified by high throughput RBP-RNA analysis.

[0136] These results demonstrated that this innovative approach can precisely inhibit functional protein-RNA interactions to accurately dissect their functions at scales and precision previously not possible.EQUIVALENTS

[0137] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within-42-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0138] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0139] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0140] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.-43-4856-2302-0756.1

Claims

Atty. Dkt. No.: 115872-3287WHAT IS CLAIMED IS1. A fusion protein comprising a PKR double-stranded RNA binding protein (RBP) domain, a nuclease-defective Cast 3 domain, and one or more subcellular-localization sequences, wherein the PKR double-stranded RBP domain comprises the amino acid sequence of AGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPE GEGRSKKEAKNAAAKLAVEILNKEKKA (SEQ ID NO: 69).

2. The fusion protein of claim 1, wherein the nuclease-defective Casl3 domain is dCasl3a, dCasl3b, or dCasl3d, optionally wherein the nuclease-defective Cast 3 domain comprises the amino acid sequence of any of SEQ ID NOs: 1-3 or 87.

3. The fusion protein of claim 1 or 2, wherein the one or more subcellular-localization sequences are selected from the group consisting of nuclear localization sequences, Endoplasmic Reticulum membrane localization sequences, mitochondrial localization sequences, cytoplasmic localization sequences, nucleolus localization sequences, and plasma membrane localization sequences.

4. The fusion protein of any one of claims 1-3, wherein the one or more subcellular- localization sequences comprise the amino acid sequences of any of SEQ ID NOs: 4- 16.

5. The fusion protein of any one of claims 1-4, comprising one subcellular-localization sequence located at the N-terminus of the nuclease-defective Cast 3 domain and the N-terminus of the PKR double-stranded RBP domain.

6. The fusion protein of any one of claims 1-4, comprising one subcellular-localization sequence located at the C-terminus of the nuclease-defective Cast 3 domain and the C-terminus of the PKR double-stranded RBP domain.

7. The fusion protein of any one of claims 1-4, comprising at least two subcellular- localization sequences.

8. The fusion protein of claim 7, wherein the at least two subcellular-localization sequences are identical or different.-44-4856-2302-0756.1Atty. Dkt. No.: 115872-32879. The fusion protein of any one of claims 7-8, wherein the at least two subcellular- localization sequences are located at the N-terminus of the nuclease-defective Casl3 domain and the N-terminus of the PKR double-stranded RBP domain.

10. The fusion protein of any one of claims 7-8, wherein the at least two subcellular- localization sequences are located at the C-terminus of the nuclease-defective Cast 3 domain and the C-terminus of the PKR double-stranded RBP domain.

11. The fusion protein of any one of claims 7-8, wherein one of the at least two subcellular-localization sequences is located at the N-terminus of the nucleasedefective Cast 3 domain and / or the N-terminus of the PKR double-stranded RBP domain, and one of the at least two subcellular-localization sequences is located at the C-terminus of the nuclease-defective Cast 3 domain and / or the C-terminus of the PKR double-stranded RBP domain.

12. The fusion protein of any one of claims 1-11, further comprising a protein tag.

13. The fusion protein of claim 12, wherein the protein tag is a biotin carboxylase carrier protein (BCCP) tag, a myc-tag, a calmodulin-tag, a FLAG-tag, a hemagglutinin (HA)- tag, a polyhistidine tag, a maltose binding protein (MBP)-tag, a nus-tag, a glutathione- S-transferase (GST)-tag, a green fluorescent protein (GFP)-tag, a thioredoxin-tag, a S- tag, a Softag, a strep-tag, a biotin ligase tag, a FlAsH tag, a V5 tag, or a SBP-tag.

14. The fusion protein of any one of claims 1-13, further comprising a detectable protein, optionally wherein the detectable protein is a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof.

15. The fusion protein of claim 14, wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

16. The fusion protein of claim 14, wherein the chemiluminescent protein is P- galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

17. The fusion protein of claim 14, wherein the fluorescent protein is TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP,-45-4856-2302-0756.1Atty. Dkt. No.: 115872-3287Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, HIKOK, mK02, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKatel, LSS- mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

18. The fusion protein of any one of claims 14-17, wherein the PKR double-stranded RBP domain and / or the nuclease-defective Cast 3 domain and the detectable protein are linked via a linker.

19. The fusion protein of claim 18, wherein the length of the linker is about 15 to about 40 amino acids.

20. The fusion protein of claim 18 or 19, wherein the linker comprises an amino acid sequence selected from the group consisting of (GGGS)n (SEQ ID NO: 70), (GGGGS)n (SEQ ID NO: 71), (G)n (SEQ ID NO: 72), (EAAAK)n (SEQ ID NO: 73), (GGS)n (SEQ ID NO: 74), (SGGS)n (SEQ ID NO: 75), SGSETPGTSESATPES (XTEN linker) (SEQ ID NO: 76), SGSETPPKKKRKVGGSPKKKRKVGTSESATPES (2X linker) (SEQ ID NO: 77), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 78), (XP)nmotif, and any combination thereof, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid.

21. The fusion protein of any one of claims 1-20, wherein the structure of the fusion protein is selected from the group consisting of:NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-COOH,NH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence] -COOH,NH2-[subcellular-localization sequence]-[nuclease-defective Casl3 domain]- [PKR double-stranded RBP domain]-[subcellular-localization sequence] -COOH,-46-4856-2302-0756.1Atty. Dkt. No.: 115872-3287NH2-[subcellular-localization sequence]-[ subcellular-localization sequence]- [nuclease-defective Casl3 domain]-[PKR double-stranded RBP domain]-COOH, andNH2-[nuclease-defective Cast 3 domain]-[PKR double-stranded RBP domain]- [subcellular-localization sequence]-[subcellular-localization sequence]-COOH, and wherein each instance of comprises an optional linker.

22. The fusion protein of any one of claims 1-21, comprising the amino acid sequence of any of SEQ ID NOs: 79-86.

23. The fusion protein of any one of claims 1-22, wherein the nuclease-defective Cast 3 domain is configured to specifically bind to a target RNA when the fusion protein is complexed with a guide RNA that is bound to the target RNA.

24. A polynucleotide comprising an open reading frame encoding the fusion protein of any one of claims 1-23.

25. The polynucleotide of claim 24, wherein the open reading frame is operably linked to an expression control sequence.

26. The polynucleotide of claim 25, wherein the expression control sequence is a tissuespecific promoter, a heterologous promoter, an inducible promoter or a constitutive promoter.

27. An expression vector or a host cell comprising the polynucleotide of any one of claims 24-26.

28. A kit comprising the expression vector of claim 27 and instructions for use, wherein the expression vector further comprises a polynucleotide that encodes a sgRNA that binds to a target RNA.

29. A kit comprising the expression vector of claim 27, a second expression vector comprising a nucleic acid that encodes a sgRNA that binds to a target RNA, and instructions for use.

30. A complex comprising (a) the fusion protein of any one of claims 1-23 and (b) a guide RNA that is configured to specifically bind to and form a duplex with a target RNA,-47-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 wherein the guide RNA is bound to the nuclease defective Casl3 domain of the fusion protein.

31. The complex of claim 30, wherein the PKR double-stranded RBP domain of the fusion protein binds to the duplex between the guide RNA and the target RNA.

32. The complex of claim 30 or 31, wherein the guide RNA comprises the nucleic acid sequence of any of SEQ ID NOs: 17-26, or 29-68.

33. The complex of any one of claims 30-32, wherein the target RNA is an oncogene mRNA, a tumor suppressor mRNA, a transcription factor mRNA, a RNA binding protein mRNA, a Ribosomal protein mRNA, a translation regulatory protein mRNA, a splicing factor mRNA, a metabolism mRNA, a pre-mRNA transcript, an intronic RNA, a Long noncoding RNA, a microRNA, a small RNA, a Ribosomal RNA, a telomerase RNA component, a promoter RNA, or an enhancer RNA.

34. A method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising a. contacting cells comprising a target RNA with the complex of any of claims 30-33 under conditions that permit binding of the complex to the target RNA, wherein the complex is configured to block binding of an RBP to a binding site within the target RNA; b. assaying the cells of step (a) for a phenotype or biomarker expression; and c. determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (a) with control cells that are not contacted with the complex and comprise the target RNA.

35. A method for identifying the effect of an interaction between a RNA binding protein (RBP) and a target RNA comprising a. transfecting cells comprising the target RNA with a polynucleotide encoding the fusion protein of any one of claims 1-23; b. transfecting the cells with a guide RNA under conditions that induce formation of a ternary complex comprising the guide RNA, the target RNA and the fusion protein, wherein the ternary complex is configured to block binding of an RBP to a binding site within the target RNA;-48-4856-2302-0756.1Atty. Dkt. No.: 115872-3287 c. assaying the cells of step (b) for a phenotype or a biomarker expression; and d. determining the effect of the interaction between the RBP and the target RNA by comparing the phenotype or biomarker expression of the cells of step (b) with control cells that are not contacted with the complex and comprise the target RNA.

36. The method of claim 35, wherein the guide RNA is configured to specifically bind to a region within the target RNA.

37. The method of any one of claims 34-36, wherein the biomarker is a genomic biomarker, a transcriptome biomarker, a proteomic biomarker, or an epigenetic biomarker.

38. The method of any one of claims 34-37, wherein the phenotype comprises viability, growth, proliferation, differentiation, translation efficiency, RNA subcellular localization, drug susceptibility or resistance, pathogen susceptibility or resistance, signal transduction, RNA stability, genomic stability, apoptosis, senescence, or cell behavior.

39. The method of any one of claims 34-38, wherein the cells are isolated from a healthy subject or a subject suffering from or diagnosed with a disease.

40. The method of claim 39, wherein the disease is cancer, an autoimmune disease, a metabolic disease or a neurodegenerative disease.-49-4856-2302-0756.1

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