Manipulating spatial RNA localization

Abstract

Described herein are localization polypeptides, RNA carrier polypeptides, polynucleotides, dimerization handles, cells, compositions, methods, and kits relating to the spatial localization of RNA in relation to a cell. In certain aspects, localization polypeptides and RNA carrier polypeptides may interact to carry an RNA of interest to a subcellular location or other location associated with a cellular membrane. In embodiments, such interaction may be covalent, for example, by the way of translational fusion, or may be inducible through a dimerization handle as described herein. In embodiments, RNA localization can be carried out on (and cells of the present disclosure can be) asymmetric cells, for example, neurons or neural progenitors. In embodiments, the ABI / PYL system can be utilized in conjunction with a catalytically-dead (i.e., nuclease-inactivated) RNA-guided CAS enzyme to precisely spatially localize an RNA of interest to a subcellular location of interest.

Description

Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC MANIPULATING SPATIAL RNA LOCALIZATION CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Patent Application No.63 / 594,147, filed October 30, 2023, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contract 2046650 awarded by the National Science Foundation and under contract CA266470, NS137219 and DK127405 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND ^

[0003] Spatial RNA transcriptomics, including subcellular (i.e., intracellular) mRNA localization, is a core mechanism for spatiotemporal regulation of gene expression and protein synthesis, which plays a critical role in diverse cellular processes and disease (Das et al., 2021). Since the first discovery in 1983 by Jeffrey et al. on the asymmetric distribution of actin mRNA in ascidian embryos using in situ hybridization (Jeffery et al., 1983), the advent of modern imaging and sequencing methods that can capture the spatial information of many RNAs, has observed thousands of RNAs that are spatially localized into distinct compartments in cells (Alon et al., 2021; Chen et al., 2015; Fazal et al., 2019). For example, neurons with large axons tend to spatially localize key mRNAs (e.g., ȕ-actin mRNA) to the tip of axon to coordinate the axon guidance and growth cone development, which presumably enhances protein production efficiency at the site of the growth cone; and reduces undesired growth of axon branches (Fernandopulle et al., 2021).

[0004] In addition, mounting evidence starts to correlate the dysregulation of mRNA localization to an increasing number of diseases. For example, neurological diseases including amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and spinal muscular atrophy (SMA) are highly correlated with aberrant mRNA spatial organization (Thelen and Kye, 2019). However, despite decades of efforts, the mechanistic and functional importance of RNA spatial 1 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC localization has only been explored for a few examples, largely due to a lack of efficient and programmable approaches that allow for the manipulation of any endogenous RNA localization in cells.

[0005] The typical experimental paradigm used to study the biological functions of RNA localization is based on engineering the genome to delete putative localization elements from RNAs and then analyzing its consequent functional influence (Buxbaum et al., 2015; Terenzio et al., 2018), which is laborious, time-consuming, and impractical to be applied broadly. In addition, genome engineering by generating stable cell lines is not feasible in most patient-derived primary tissues, and it also influences properties including epigenetics, splicing, translation, and RNA stability. Furthermore, the lack of strategies to manipulate the localization of any endogenous RNAs severely hinders the understanding of the mechanisms behind the diseases correlated with abnormal spatial RNA localization and even the development of potential therapeutics.

[0006] Current state-of-the-art strategies to manipulate RNA localization can be classified into three types:

[0007] The first one is based on the removal of localization elements from RNAs to disturb its correct subcellular localization (Miller et al., 2002; Terenzio et al., 2018; Yoon et al., 2012). For example, to directly test the functional role of dendritic localization of CAMK2A mRNA, its 3’UTR which mediates mRNA localization to dendrites was removed and the protein level of CAMK2A and neuron functions were analyzed (Miller et al., 2002). However, this strategy is difficult to be widely applied since the localization elements of most RNAs are currently unknown and it is laborious and time-consuming to create cell lines lacking these localization elements. In addition, the removal of localization elements may influence key properties (translation, stability, etc.) of the target RNA.

[0008] The second type of strategies tags target transcripts with RNA aptamers (such as MS2 and TetR-binding aptamers) and fuses aptamer-binding proteins with proteins natively involved in localizing endogenous transcripts for repositioning (Belmont and Niles, 2012; Katz et al., 2012). For example, MS2 stem-loops were encoded inside the 3’UTR of ȕ-actin mRNA which was tethered to focal adhesions by MS2 coat protein (MCP)-vinculin fusion proteins, leading to the increase of adhesion size and lifetimes (Katz et al., 2012). However, this type of strategy requires prior tagging of target transcripts with RNA aptamers which is time-consuming for endogenous 2 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC transcripts and may also influence key properties (translation, stability, etc.) of the target RNA. The strategy does not work for primary cells or in vivo.

[0009] The third one utilized a PUF (Pumilio and FBF homology domain)-assisted localization of RNA (PULR) system, which recognizes endogenous mRNAs by the PUF domains that bind to specific RNA codes, to reposition mRNAs to the cellular periphery or the perinuclear region (Abil et al., 2017). However, reprogramming PUF domains is laborious, and the PUF domain often suffers from its off-target binding to its natural mRNA targets within cells. SUMMARY

[0010] In embodiments, described herein are cells comprising a heterologous intracellular molecular transport dimerization pair. In embodiments of a cell described herein, the pair can comprise a localization polypeptide, and a ribonucleic acid (RNA) carrier polypeptide.

[0011] In embodiments, the localization polypeptide can comprise an amino acid sequence encoding a subcellular compartment polypeptide or a motor polypeptide. In embodiments, the subcellular compartment polypeptide or motor polypeptide can comprise a telomere polypeptide, a mitochondrial polypeptide, a endoplasmic reticulum polypeptide, a lysosomal polypeptide, a p- body polypeptide, a stress granule polypeptide, a TIS granule polypeptide, a peroxisomal polypeptide, a ribosomal polypeptide, a golgi apparatus polypeptide, a cytoskeletal polypeptide, a cytosolic polypeptide, a vacuole polypeptide, a cellular membrane polypeptide, an exosomal polypeptide, a nuclear polypeptide, a nucleolar polypeptide, a cytoplasmic polypeptide, or a plasma membrane polypeptide.

[0012] In embodiments, the motor polypeptide can comprise a myosin polypeptide, a kinesin superfamily polypeptide, a dynein polypeptide, a polymerization motor polypeptide, a rotary motor polypeptide, or a nucleic acid motor polypeptide. In embodiments, the localization polypeptide can encode a mitochondrial antiviral signaling polypeptide (MAVS), a translocase of the outer membrane (TOM) polypeptide, a ras GTPase-activating polypeptide-binding polypeptide 1 (G3BP1) polypeptide, a DEAD-Box Helicase 6 (DDX6) polypeptide, a truncated kinesin family member C1 (KIFC1), a truncated kinesin family member 5a (KIF5a), or a truncated kinesin family member 5b (KIF5b).

[0013] In embodiments, the RNA carrier polypeptide can comprise an amino acid sequence encoding a dead CRISPR-associated (dCas) polypeptide. In embodiments, the dCas polypeptide 3 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC can be a type II, type IV, type III, or type VI CRISPR system dCas polypeptide. In embodiments, the dCas polypeptide is a dCas13 polypeptide or dCas7-11 polypeptide. In embodiments, the dCas13 is a dCas13a polypeptide, dCas13b polypeptide, dCas13c polypeptide, or dCas13d polypeptide. In embodiments, a cell can further comprisie a guide RNA (gRNA) configured to specifically bind to (a) the RNA carrier polypeptide and (b) an intracellular RNA. In embodiments, the intracellular RNA can comprise a polynucleotide encoding GAPDH, ACTB, SDHC, SDHD, NDUFS2, Psd95, CamKIIa, Grin2b, CD47, BCL-2, CD274 / PDL1, p53, EGFR, GPCR, or TFRC / CD71.

[0014] In embodiments, the localization polypeptide and RNA carrier polypeptide can be translationally fused. In embodiments, the localization polypeptide can further comprise a first dimerization handle translationally fused with the subcellular compartment polypeptide or a motor polypeptide, and the RNA carrier polypeptide comprises a second dimerization handle translationally fused with the RNA carrier polypeptide, wherein the first and second dimerization handles bind to one another.

[0015] In embodiments, the first and second dimerization handle can be chemically or optically inducible. In embodiments, the first dimerization handle can comprise a pyrabactin resistance 1- like (PYL) polypeptide, a first FKBP12 polypeptide, or a FKBP polypeptide; and b. the second dimerization handle can comprise an ABI polypeptide, a second FKBP12 polypeptide, a FRB polypeptide, a calcineurin polypeptide, or a cyclosporin A polypeptide. In embodiments, a. the first dimerization handle can comprise an ABI polypeptide, a second FKBP12 polypeptide, a FRB polypeptide, a calcineurin polypeptide, or a cyclosporin A polypeptide; and b. the second dimerization handle can comprise a PYL polypeptide, a first FKBP12 polypeptide, or a FKBP polypeptide, respectively. In embodiments, the PYL polypeptide is an Arabidopsis thaliana PYL1 polypeptide. In embodiments, the ABI polypeptide is an Arabidopsis thaliana ABI polypeptide.

[0016] In embodiments, a cell can further comprise a first detectable label linked to the localization polypeptide or the RNA carrier polypeptide. In embodiments, a cell can further comprise a second detectable label linked to the localization polypeptide, or the RNA carrier polypeptide not linked to the first detectable label. In embodiments, a cell can further comprise abscisic acid (ABA), causing the binding of (a) and (b) within the cell.

[0017] In embodiments, a localization polypeptide can be linked to the RNA carrier polypeptide by a linker. 4 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0018] In embodiments, the cell is an animal cell. In embodiments, the cell is not a plant cell. In embodiments, a cell is a neuron, a glial cell, a fibroblast, an intestinal epithelial cell, a mesenchymal cell, a T cell, a cancer cell, a stem cell, or a cell from an immortalized cell line.

[0019] Also described herein are methods of localizing RNA in a cell. In embodiments, a method of localizing RNA in a cell can comprise delivering nucleic acids encoding a localization polypeptide and RNA carrier polypeptide as described herein into a cell and inducing dimerization of the localization polypeptide and RNA carrier polypeptide inside the cell, forming an intracellular molecular transport dimerization pair. In embodiments, methods as described herein can further comprise delivering a guide RNA to the cell. In embodiments, the inducing is optical or chemical induction. In embodiments, the inducing is by delivering abscisic acid to the cell.

[0020] In embodiments of methods as described herein, the cell can be an animal cell. In embodiments of methods as described herein, the cell is not a plant cell.

[0021] In embodiments, methods as described herein can further comprise detecting the dimerization pair.

[0022] Also described herein are nucleic acid constructs. In an embodiment, a nucleic acid can encode a localization polypeptide as described herein. In embodiments, a nucleic acid can encode a ribonucleic acid (RNA) carrier polypeptide as described herein.

[0023] Also described herein are vectors. In embodiments, a vector can comprise any nucleic construct as described herein. Methods of using vectors as described herein are also described. In embodiments, vectors can be delivered to a cell by any suitable method for introducing DNA into a cell as described herein (for example, lipofection, electroporation, viral transduction, and the like). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0025] FIG.1 is an illustration of an inducible and programmable CRISPR-mediated transcript organization (CRISPR-TO) system for repositioning RNAs to various desired subcellular compartments. dCas13 protein is fused with ABI (hereafter referred to as dCas13-ABI) and the 5 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC cognate heterodimer partner PYL1 is fused with a compartment-specific protein or a motor protein. After adding the ABA ligand, dCas13 will recruit the target RNA to the chosen subcellular compartment.

[0026] FIGs. 2A-2C. CRISPR-TO manipulates the spatial localization of repetitive reporter mRNAs efficiently. FIG. 2A shows the representative microscopic images showing the localization of GCN4x24 reporter mRNAs to OMM in HeLa cells in the gGCN4+ABA group, but not in the other two groups. Scale bars, 20 μm. gGCN4 is the gRNA targeting the GCN4x24 reporter mRNA. gNT is the non-targeting gRNA. FIG. 2B shows the box-and-whisker plots depicting the percentage of dCas13-ABI and GCN4x24 reporter mRNA localized on OMM per cell quantified from 51-57 cells per group. FIG. 2C shows the box-and-whisker plots depicting the percentage of different reporter mRNAs localized on OMM per cell quantified from 52-60 cells per group after ABA treatment. n.s., not significant (p > 0.05); **p < 0.01; ***p < 0.001, unpaired t-test between gNT and indicated groups. For all the quantifications in this figure, only analyze cells co-expressing dCas13-ABI, MAVS*-PYL1, and reporter mRNAs. For all box-and- whisker plots in this patent application, the boxes show 25% to 75% with the bar indicating the median value and the whiskers extend to 10% and 90%, with individual data points shown for the lowest and highest 10% of each dataset.

[0027] FIGs. 3A-3C. CRISPR-TO manipulates the spatial localization of endogenous mRNAs efficiently. FIG. 3A shows the representative microscopic images showing the localization of GAPDH mRNAs to OMM in HeLa cells in the gG123+ABA group, but not in the two control groups. Scale bar, 20 μm. gG123 represents the mixture of three gRNAs (gG1, gG2, and gG3) targeting GAPDH mRNA. FIG.3B shows the box-and-whisker plots depicting the percentage of dCas13-ABI and GAPDH mRNA localized on OMM per cell quantified from 59-70 cells per group. FIG. 3C shows the box-and-whisker plots depicting the percentage of GAPDH mRNA localized on OMM per cell after ABA treatment for cells transfected with dCas13-ABI, MAVS*- PYL1, and gNT or different combinations of three GAPDH gRNAs. 36-70 cells were quantified per group. n.s., not significant (p > 0.05); **p < 0.01; ***p < 0.001, unpaired t-test.

[0028] FIGs. 4A-4B. Schematic of different subcellular compartments and the corresponding plasmids of PYL1-fusion proteins. FIG.4A shows the schematic of plasmids expressing different PYL1-fusion proteins for localizing mRNA to different subcellular compartments. FIG.4B shows a schematic cell showing seven different subcellular compartments to which mRNA can be 6 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC repositioned by CRISPR-TO. To reposition mRNA to outer mitochondrial membrane (OMM), p- bodies, and stress granules, which are cytoplasmic locations, PYL1 was fused with the C-terminal 31 amino acids of mitochondrial antiviral-signaling protein (MAVS*), DDX6 protein, and G3BP1 protein, respectively. To reposition mRNA to telomere and nuclear stress bodies which are nuclear locations, PYL1 was fused with TRF1 protein and HSF1 protein, respectively. To transport mRNA toward the plus ends of microtubules, PYL1 was fused with a truncated kinesin motor protein Kif5a(aa1-559) (named as KIF5a*) or KIF5b(aa1-555) (named as KIF5b*). To transport mRNA toward the minus ends of microtubules where the centromeres are located, PYL1 was fused with a truncated kinesin motor protein KIFC1(aa125-673) (named as KIFC1*).

[0029] FIG.5 shows the representative microscopic images showing the localization of GAPDH mRNAs or GCN4x24 reporter mRNAs to p-bodies, stress granules, minus ends of microtubules, plus ends of microtubules, telomere, and nuclear stress bodies in HeLa cells after CRISPR-TO perturbation. The shape of the nucleus was depicted with a white dotted line in some cells. Scale bar, 20 μm.

[0030] FIGs. 6A-6E. CRISPR-TO is a chemically inducible and reversible system. FIG. 6A shows the box-and-whisker plots depicting the percentage of dCas13-ABI and GAPDH mRNA localized on OMM per cell after treating HeLa cells transfected with dCas13-ABI, MAVS*-PYL1, and gG123 with different concentrations of ABA for 4 hours.27-50 cells were quantified for each group. FIG. 6B shows the box-and-whisker plots depicting the percentage of dCas13-ABI and GAPDH mRNAs localized on OMM per cell after treating HeLa cells transfected with dCas13- ABI, MAVS*-PYL1, and gG123 with 250 μM ABA for different periods. FIG.6C shows the box- and-whisker plots depicting the percentage of dCas13-ABI and GAPDH mRNAs localized on OMM per cell after treating HeLa cells transfected with dCas13-ABI, MAVS*-PYL1, and gG123 with 250 μM ABA for 4 hours followed by removing ABA for different periods. The right column of DMSO represents the same transfected cells but treated with DMSO for 4 hours. 26-50 cells were quantified for each group. FIG. 6D shows the schematic of the MS2-mRNA applied for the dynamic tracking of mRNA repositioned by CRISPR-TO in live cells. U2OS-2-6-3 cells were transduced with lentivirus expressing rtTA and stdMCP-tdTomato, followed by electroporation to deliver CRISPR-TO components (dCas13-ABI, MAVS*-PYL1, and gRNA targeting MS2) for repositioning and dynamic tracking of MS2-mRNA. FIG.6E shows the distance to the centromere of five representative type I trajectories (confined, sub-diffusive motion) and type II trajectories 7 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC (super-diffusive motion) as a function of time. n.s., not significant (p > 0.05); **p < 0.01; ***p < 0.001, unpaired t-test between 0 μM and indicated groups.

[0031] FIGs. 7A-7C. CRISPR-TO promotes the localization of endogenous mRNA in the projections of primary neurons. FIG.7A shows the schematic of applying CRISPR-TO to promote the localization of mRNA in the neuronal projections. ACTB mRNA is transported toward the plus end of microtubules by truncated Kif5a (Kif5a*). FIG.7B shows the representative confocal images of a primary mouse hippocampal neuron in the gT+ABA group showing ACTB mRNAs enriched at the terminals of neuronal projections and inside neuronal projections after CRISPR- TO perturbation. gT represents gRNAs targeting ACTB mRNA. The left image shows a neuron with all four channels merged. A black rectangle was put in the back of the tiled images. Scale bar, 100 μm. Right: magnification of dotted boxes in the left image. Scale bar, 50 μm. FIG.7C shows the scatter plot of the fluorescence intensity of ACTB mRNA in the terminals of neuronal projections. Each dot represents one terminal of one neuronal projection, and 128-178 terminals were quantified for each group. The black line represents the median value of the distribution. ***p < 0.001, unpaired t-test.

[0032] FIGs.8A-8D. The mRNA recruited to the terminals of neuronal projections by CRISPR- TO in primary mouse hippocampal neurons can be translated locally to produce proteins. FIG.8A shows the schematic of the plasmid expressing NLS-HaloTag-GCN4x24 reporter mRNA with 24 GCN4 repeats in its 3’UTR. FIG. 8B shows the Schematic of detecting the local translation of NLS-HaloTag-GCN4x24 reporter mRNAs transported to the projection terminals by CRISPR-TO in mouse hippocampal neurons. FIG. 8C shows the representative microscopic images showing the amount of reporter proteins in the projection terminals of gGCN4+ABA group is much higher than that of gGCN4+DMSO and gNT+ABA groups. Scale bar, 100 μm. Right: insets of dotted boxes. Scale bar, 50 μm. Calibration bar indicates the fluorescence intensity of reporter proteins. FIG.8D shows the box-and-whisker plots depicting the ratio of fluorescence intensity of reporter proteins in the projection terminal divided by that in the soma quantified from 42-71 projection terminals per group.

[0033] FIGs.9A-9E. The axonal localization of ACTB mRNA influences the growth of axons. FIG. 9A shows the representative microscopic images showing the localization of ACTB mRNA and dCas13-ABI, and the shape of Neuro-2a-differentiated neurons in three groups. Scale bar, 50 μm. FIG. 9B shows the box-and-whisker plots depicting the fluorescence intensity of ACTB 8 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC mRNA in one axonal tip of Neuro-2a-differentiated neurons in three groups. 104-126 axonal tips were quantified for each group. FIG. 9C shows the box-and-whisker plots depicting the fluorescence intensity of ACTB mRNA per μm axon of Neuro-2a-differentiated neurons in three groups. 90-100 axons were quantified for each group. FIGs. 9D-9E show the box-and-whisker plots depicting the axon length of Neuro-2a-differentiated neurons in different groups. 110-195 axons were quantified for each group. n.s., not significant (p > 0.05); *p < 0.05; ***p < 0.001, unpaired t-test.

[0034] FIGs.10A-10D. OMM localization of mRNA promotes the import of translated proteins into mitochondria. FIG.10A shows the schematic of three reporter mRNAs. MTS, mitochondrial targeting sequence. DHFR acts as degrons. FIG. 10B shows the quantification of the median intensity of mRuby3 under different conditions after recruiting different reporter mRNAs to OMM using MAVS*-PYL1 by CRISPR-TO. FIG.10C shows the quantification of the median intensity of mRuby3 under ABA or DMSO treatment after recruiting Reporter-3 mRNA to OMM using MAVS*-PYL1, TOM22-PYL1, TOM20-PYL1, or TOM70-PYL1 by CRISPR-TO. Data presented as means ± SD. n = 3 (n represents the number of transfection replicates). n.s., not significant (p > 0.05); *p < 0.05; **p < 0.01, unpaired t-test. FIG. 10D shows a model of OMM localization of mRNA promoting the import of translated proteins into mitochondria. The MTS- Protein-DHFR translated from mRNAs diffused in the cytoplasm is quickly degraded before it is imported into the mitochondria. Localizing mRNAs to OMM by CRISPR-TO promotes the import of translated proteins into the mitochondria, protecting proteins from being degraded by the proteasomes in the cytoplasm. DETAILED DESCRIPTION

[0035] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included. 9 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 1. ABBREVIATIONS AND DEFINITIONS

[0036] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0037] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0038] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements.

[0039] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0040] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term 10 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0041] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 11 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC mixed-base and / or deoxyinosine residues. Polynucleotides as described herein may further comprise detectable labels (such as reporters and affinity purification agents) such as a fluorophore, repeated sequences (polyA sequences, for example) and the like. In embodiments, a polynucleotide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to a reference polynucleotide. For example, a PYL1 polynucleotide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to a polynucleotide encoding Arabidopsis thaliana protein_id="NP_001331305.1” (SEQ ID NO:1).

[0042] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0043] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. Non-viral methods can involve creating transient pores in the cellular membrane that allow for passive (i.e., transfection) or active (i.e., electroporation) diffusion of nucleic acids into a cell. The nucleic acid molecules may be gene sequences encoding complete proteins or can be truncated, encoding only the necessary functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation (which utilizes an electric current to transiently and reversibly porate the cellular membrane, which can maintain cellular viability while the current also drives net flux of charged molecules, such as nucleic acids, into a cell) following standard procedures well known in the art. For viral-based methods of transfection any useful lentiviral vector may be used in the methods described herein. Without intending to be limiting, examples of viral vectors include, lentiviral viral vectors, which can be utilized to form a lentivirus capable of introducing genetic material into a cell. Lentiviral vectors can also be driving into a cell using standard methods such as those described above (transfection, for example). In some embodiments, the nucleic acid molecules are introduced into a cell using a lentiviral vector following standard procedures well known in the art. In embodiments, for example, after the 12 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC packing and formation of lentivirus particles, the functional lentiviral particles can then infect a host cell via lentiviral transduction, thereby introducing polynucleotides into the cell that had been packaged in the virus. The terms ƎtransfectionƎ or ƎtransductionƎ also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0044] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0045] Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression" the transfected gene is not transferred to the daughter cell during cell division, and such expression may not be constitutive (i.e., may be able to switched on and off using an inducible system such as a Tet-On or Tet-Off system, which allows for transgene expression to be switched on or off in the presence or absence of an antibiotic or other molecular “switch”). Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast to transient expression in which the transfected gene is not passed down to daughter cells, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell and allows for the transfected gene to be passed down to subsequent generations. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Expression of a transfected gene, in particular stable expression, can further be accomplished by transposon-mediated insertion into to the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision.

[0046] The term "plasmid" refers to a nucleic acid molecule (i.e., a polynucleotide) that encodes for genes and / or regulatory elements necessary for the expression of genes in the presence of the requisite transcriptional and translational machinery. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by 13 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0047] The term “exogenous” refers to a molecule or substance (e.g., nucleic acid or protein) that originates from outside a given cell or organism. Conversely, the term “endogenous” refers to a molecule or substance that is native to, or originates within, a given cell or organism. The skilled artisan would understand that the introduction of an exogenous substance into the cell according to the present disclosure will result in a heterologous polynucleotide or polypeptide that is introduced into the cell (i.e., the cell would then harbor both the endogenous and exogenous variants, if the exogenous polynucleotide or polynucleotide is also present endogenously in the cell, where the exogenous variant may be a recombinant, truncated, mutated, or other version of the endogenous polynucleotide or polypeptide that may not be naturally-occurring).

[0048] The term "vector" refers to a carrier DNA molecule (i.e., a polynucleotide) into which a DNA sequence can be inserted for introduction into a host cell. In some embodiments, vectors of use according to the present disclosure are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector, e.g., a promoter. Vectors include non-viral vectors such as plasmids and viral vectors (for example lentiviral and adenoviral vectors). Vectors as described herein additionally may be capable of self-replication and may be able to be passed down from a cell to its daughter progeny.

[0049] A “viral vector” is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include lentiviral vectors.

[0050] The term “operably linked” refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term “operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of 14 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence. In certain embodiments, polypeptides as described herein may be “operably linked” such that two different polypeptides are transcribed from a single continuous nucleic acid sequence, or otherwise translationally fused, ultimately leading to the production of a fusion protein (a localization / RNA carrier fusion polypeptide, for example).

[0051] The terms "regulatory sequence" and "promoter" are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein. In some instances, the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.

[0052] "Expression cassette" refers to a polynucleotide comprising a promoter or other regulatory sequence operably linked to a sequence encoding a protein.

[0053] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain, wherein each amino acid residue is linked to another by a peptide bond unless otherwise specified. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct. The term “truncated” refers to a portion of a polypeptide that is not a full-length polypeptide when compared to a reference sequence and comprises at least one or more functional elements of that 15 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC polypeptide. For example, a “truncated” motor polypeptide comprises the localization domain that is necessary to target a localization polypeptide to the proper subcellular location, in addition to any domains necessary for movement along a portion of the cytoskeleton to which it is target, but omits other polypeptide sequences comprising elements of the motor protein not required for this functionality. Polypeptides as described herein may further comprise detectable labels (such as reporters and affinity purification agents) such a fluorophore, repeated sequences (His-tag sequences, for example) and the like. In embodiments, a polypeptide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to a reference polypeptide. For example, a PYL1 polypeptide as described herein can be at least 80, 85, 90, 95, 98%, or 99% identical to Arabidopsis thaliana protein_id="NP_001331305.1” (SEQ ID NO:1).

[0054] The term “amino acid” refers to naturally occurring and synthetic 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 occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, Ȗ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an Į carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0055] Amino acids may be 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, may be referred to by their commonly accepted single-letter codes.

[0056] The term “a recombination of amino acid sequences,” in the context of a peptide, refers to a change or variation in the amino acid sequence of a reference peptide, such that the biological properties of the reference peptide are maintained after the amino acid sequence change. For example, the recombination of amino acid sequence may be a conservative amino acid substitution 16 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC or an amino acid sequence modification (addition, deletion, or substitution) to produce a chimeric peptide.

[0057] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581-87 (2013); Xie et al. “Adding amino acids to the genetic repertoire,” Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0058] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post- translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0059] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0060] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity 17 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC method of Pearson & Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0061] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-10 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation I of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0062] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. ’at'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the 18 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0063] As used herein, “localization polypeptide” refers to a polypeptide capable of translocating a cargo (e.g., a bound polypeptide and / or nucleic acid) from one physical location inside a cell (or along the exterior cellular membrane) to a second physical location (i.e., a target location or a target) that is different than the first physical location. Localization polypeptides as described herein can comprise an amino acid sequence capable of binding to a target (i.e., a subcellular compartment polypeptide or motor polypeptide as further discussed below), for example, a polypeptide of the cellular membrane, a motor protein, a subcellular compartment (such as the outer mitochondrial membrane, for example), or a cytoskeletal component. Localization polypeptides as described herein can also comprise an amino sequence capable of binding a cargo (for example, another polypeptide or a polynucleotide) such that binding of the cargo to the localization polypeptide causes the cargo to be translocated with the localization peptide to a target location. Such binding may be covalent or non-covalent, and can be direct (i.e., localization polypeptide to cargo) or may be indirect and occur through a linker or other intermediary (for example, a small molecule intermediary, a polynucleotide intermediary, or a polypeptide intermediary).

[0064] As used herein, “ribonucleic acid (RNA) carrier polypeptide” refers to a polypeptide capable of binding an RNA for transport along a cellular membrane or within a cell. In embodiments of RNA carrier polypeptides as described herein, such polypeptides can bind to, or otherwise interact with, localization polypeptides as described herein as the “cargo” of the localization peptide. Such binding or interaction with the localization polypeptide causes translocation of the RNA carrier polypeptide along with the localization polypeptide, along with any RNA bound to the RNA carrier polypeptide.

[0065] As used herein, “subcellular compartment polypeptide” refers to a polypeptide associated with (i.e., one that binds to, integrates with, and the like) an intracellular compartment. Such intracellular compartment may be a distinct cellular structure, for example, the membrane of an organelle, the cellular membrane, or cytoskeleton. Subcellular compartments may also refer to local regions inside a cell with distinct physical characteristics and properties that can contain higher concentrations of a polypeptide that localization polypeptides as described herein may bind to. 19 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0066] As used herein, “motor polypeptide” refers to a polypeptide that can transform cellular energy (such as that derived from hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP), for example) into motion or mechanical work. Localization polypeptides as described herein can utilize such motor polypeptides for active translocation within in a cell, for example by binding to a kinesin or dynein, which would then allow for transport along cytoskeletal elements with an energy input to the motor polypeptide. 2. INTRODUCTION

[0067] Methodologies to precisely manipulate the location and spatial information of RNAs, including mRNA or noncoding RNAs, can greatly strengthen the potential of mRNA-based therapies. Such methods can allow one to precisely control when and where the mRNA will be translated, or noncoding RNAs (such as microRNA, long noncoding RNA, or circular RNA) to exert their regulatory functions. Furthermore, such technologies can help control the effects from RNA therapies in larger cells, including neurons, intestinal epithelium, embryos, or at the interface of neuromuscular junction.

[0068] Methods described herein relate to subcellular RNA manipulation. In some embodiments, the method is referred to as CRISPR-Mediated Transcript Organization (CRISPR-TO; FIG. 1). The methods can allow for the precise manipulation of any endogenous RNA localization with high temporal and spatial precision using CRISPR technology.

[0069] The methods described herein comprise localizing one or more RNAs to a particular subcellular location as targeted by a localization polypeptide. For example, in some embodiments, an RNA carrier polypeptide binds to a target RNA and is subsequently localized to a subcellular location by associating with a localization polypeptide that carries the RNA carrier polypeptide and bound RNA to a subcellular location to which the localization polypeptide binds. As explained below, there are various configurations of both the RNA carrier polypeptide and localization polypeptide as well as how to induce binding of the RNA carrier polypeptide to the localization polypeptide as described in detail below. Any or all of the components described herein, e.g., the localization polypeptide and the RNA carrier polypeptide, gRNAs, or other components, can be heterologous or exogenous to the cell in which they occur, i.e., they may be components that are not present in a cell of interest under normal physiological conditions or components. 20 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 2.1. Subcellular Localization

[0070] Subcellular localization can be achieved by expressing or otherwise delivering a localization polypeptide in or to the cell and allowing the localization polypeptide to bind to its target at a subcellular location before, during or following association with the RNA carrier polypeptide (i.e., dimerization of the localization polypeptide and RNA carrier polypeptide). As discussed elsewhere in the present disclosure, the association of the localization peptide with the RNA carrier polypeptide may occur between the two polypeptides directly based on amino acid sequences having affinity to one another, may occur through an intermediary (such as a small molecule, polypeptide, or nucleic acid linker), or the association may be a covalent one in the form of a single polypeptide transcribed from a single nucleic acid sequence (i.e., a fusion protein). An RNA of interest can then be translocated upon binding of the RNA to the RNA carrier polypeptide, which is then translocated within a cell upon binding of the localization polypeptide to its target.

[0071] Exemplary subcellular locations according to the present disclosure which RNA can be translocated to or from can include but are not limited to: cellular nucleoplasm; the cellular nucleolus, for example, the fibrillar center, the dense fibrillar component, or the granular component; the nucleus, including any component of the nuclear matrix and / or the inner or outer nuclear membrane; cellular locations where chromatin may be found; ribosomes associated with any subcellular membrane; a telomere of nuclear chromosomes; subcellular vesicles (for example, vacuoles, lysosomes, transport vesicles, and secretory vesicles, such as synaptic vesicles in neurons), including inner and outer vesicular membranes; the outer mitochondrial membrane; the inner mitochondrial membrane; mitochondrial cristae; the mitochondrial matrix; the rough endoplasmic reticulum; the smooth endoplasmic reticulum; cisternae of the endoplasmic reticulum; sarcoplasmic reticulum; ribosomes of the rough endoplasmic reticulum; transport vesicles; the trans- face of the golgi apparatus; the cis- face of the golgi apparatus; the cisternae of the golgi apparatus; golgi bodies; the inner membrane, outer membrane, or lumen of a lysosome; a centrosome; cytoskeletal structures, for example microfilaments, microtubules, and intermediate filaments; vacuoles; and intracellular cytosolic areas where polypeptides of interest may be present in a greater concentration than other cytosolic areas, for example, in membrane-less stress granules.^ 21 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 2.2. Localization Polypeptides

[0072] Described herein are localization polypeptides that aid in the intracellular translocation of RNA from one physical point in a cell to another. It will be understood that a localization polypeptide of a particular cellular structure as described herein allows for localization to that cellular structure. In some embodiments, the localization polypeptide comprises a subcellular compartment polypeptide (i.e., a polypeptide present in or integrated with a cellular compartment) or a motor polypeptide that allows for targeting of the localization polypeptide to a physical region inside a cell. In some embodiments, the localization polypeptide is a subcellular compartment polypeptide or a motor polypeptide. In some embodiments, the localization polypeptide may comprise a polypeptide that can irreversibly or reversibly bind to other otherwise interact with a subcellular compartment polypeptide or a motor polypeptide as described herein (for example, an antibody whose epitope is a subcellular compartment polypeptide or a motor polypeptide as described herein).

[0073] In some embodiments, the localization polypeptide is a subcellular compartment polypeptide or a motor polypeptide that can interact with (or be modified to interact with) an RNA carrier polypeptide. In some embodiments, the localization polypeptide may also comprise an exogenous polypeptide that is not otherwise present in the cell under normal physiological conditions (for example, an Arabidopsis thaliana PYL1 polypeptide, which is a plant polypeptide that can be translated in a mammalian cell from a vector according to embodiments of the present disclosure and further described in subsequent sections, or other recombinant or truncated polypeptide having an amino acid sequence that itself is not endogenously present inside a cell of interest) that may be translationally fused with a subcellular compartment protein or motor protein. In other embodiments, the localization polypeptide may also bind to and translocate RNA directly, or otherwise be covalently bound to (and therefore translationally fused with) an RNA carrier polypeptide.

[0074] In embodiments, a localization polypeptide can be a nuclear polypeptide. In embodiments, the nuclear polypeptide can be a Lamin A / C or proliferating cell nuclear antigen (PCNA). In embodiments, a localization polypeptide can be a mitochondrial polypeptide. In embodiments, the nuclear polypeptide can be a cytochrome C or Tom20 polypepide. In embodiments, a localization polypeptide can be an endoplasmic reticulum polypeptide. In embodiments, an endoplasmic reticulum polypeptide can be a calnexin or Sec61 polypeptide or 22 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC N-terminal 27 amino acids of endoplasmic reticulum (ER)-resident protein P450 oxidase 2C1 for outer ER membrane. In embodiments, a localization polypeptide can be a golgi apparatus polypeptide. In embodiments, a golgi apparatus polypeptide can be a GM130 or TGN46 polypeptide. In embodiments, a localization polypeptide can be a lysosomal polypeptide. In embodiments, a lysosomal polypeptide can be a lysosome-associated membrane protein 1 (LAMP1) polypeptide. In embodiments, a localization polypeptide can be a peroxisomal polypeptide. In embodiments a peroxisomal polypeptide can be a PEX14 or ABCD1 polypeptide. In embodiments, a localization polypeptide can be a plasma membrane polypeptide. In embodiments, a plasma membrane polypeptide can be an EpCAM polypeptide, or intracellular fragment of a transmembrane receptor protein. In embodiments, a localization polypeptide can be a lysosomal polypeptide. In embodiments, a lysosomal polypeptide can be an actin or tubulin. In embodiments, the localization polypeptide can be an inner or outer cell membrane polypeptide. In embodiments, the inner cell membrane polypeptide can be CAAX or myr. In embodiments, the localization polypeptide can be an inner or outer nuclear membrane polypeptide. In embodiments, the outer nuclear membrane polypeptide can be KASH4 or a KASH4 domain.

[0075] In embodiments, a localization polypeptide can be a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to any one of SEQ ID NOs:3-14. 2.2.1 Subcellular Compartment Polypeptides

[0076] Without intending to be limiting, examples of subcellular compartment polypeptides according to the present disclosure include: a telomere polypeptide (for example, one or more of repressor / activator protein 1 (RAP1), telomeric repeat-binding factor 1 (TFR1), telomeric repeat- binding factor 2 (TRF2), TERF1-interacting nuclear factor 2 (TIN2), POT1-interacting protein 1 (TPP1), and / or protection of telomere (POT1)); a mitochondrial polypeptide (such as a mitochondrial antiviral signaling polypeptide (MAVS) or a translocase of the outer membrane (TOM) polypeptide, for example, TOM20, or cytochrome C); a smooth or rough endoplasmic reticulum polypeptide (for example, one or more of sarcoplasmic / endoplasmic reticulum Ca2+- ATPase (SERCA2), calreticulin, collagen, type I, alpha 1 (COL1A1), calnexin, heme oxygnase 1 (HO-1), heme oxygnase 2 (HO-2), (ryanodine receptor 1 (RyR1), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), and / or Sec61); a lysosomal polypeptide (for example, lysosomal- associated membrane protein 1 (LAMP1) or lysosomal-associated membrane protein 1 (LAMP- 2)); a p-body polypeptide or a stress granule polypeptide (for example, a DEAD-Box Helicase 6 23 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC (DDX6) polypeptide)); a TIS granule polypeptide (for example, a 12-O-tetradecanoylphorbol-13- acetate (TPA) inducible sequence 11 (TIS11) polypeptide); a peroxisomal polypeptide (for example, catalase or peroxisomal membrane protein 70 (PMP70); a ribosomal polypeptide (for example, ribosomal proteins L7a, L26 (a component of the 60S subunit), S3, S6, S10, S11 (40S subunit); a trans- or cis-golgi apparatus or golgi body polypeptide (for example, one or more of a formiminotransferase cyclodeaminase (FTCD) such as 58K, estrogen receptor binding site associated antigen 9 (EBAG9), Golgi matrix protein 130 (GM130), trans-golgi network protein 2 (TGN46), component of oligomeric golgi complex 2 (COG2), syntaxin 6, and / or golgi glycoprotein 1 (GLG1)); a cytoskeletal polypeptide (for example, actin, tau, vimentin, calretiulin, inducible nitric-oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS)); a cytosolic polypeptide (for example, a stress granule polypeptide such as ras GTPase-activating polypeptide- binding polypeptide 1 (G3BP1) polypeptide); a vacuole polypeptide (for example, a class I or class II veli-associated membrane-associated guanylate kinase (MAGUK) 1 (vam) protein); a cellular membrane polypeptide (for example, an ionotropic membrane-bound receptor or subunit thereof (such as glutamate receptor 1 (GluR1), a subunit of Į-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARS), for example) or a metabotropic receptor or subunit thereof (for example metabotropic glutamate receptor subtype 5 (mGluR5)), cadherin, a protein of the CD98 complex, and / or plasma membrane calcium ATPase (PMCA)); a nuclear polypeptide (for example, a histone (such as histone H4, H2A.X, or H2A.Z), a hypoxia-inducible factor 1-alpha (HIF1A or HIF1Į); a nucleolus polypeptide (for example, fibrillan or another nucleolar protein (NOP) family protein such as three tandem nucleolar targeting sequences from NF-kB-inducing kinase (NIK3x) for nucleolus); a centrosomal polypeptide and the like (for example a gamma tubulin, ninein, and / or polo-like kinase 4 (PLK4)). A subcellular compartment polypeptide can be chosen according to a cell type of interest or a location of interest.

[0077] In other embodiments, localization polypeptides as described herein may comprise a polypeptide that is capable of reversibly or irreversibly binding to or otherwise non-covalently interacting with a subcellular compartment polypeptide or motor polypeptide as described above. Without intending to be limiting, examples of which include one or more polypeptides having a complementary sequence to a subcellular compartment polypeptide or motor polypeptide as described herein that may bind to a polypeptide sequence of interest (for example, a polypeptide comprising one or more complementary determining-regions (CDRs) to a subcellular 24 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC compartment polypeptide or motor polypeptide as described herein, such as a heavy-chain variable region of an immunoglobin that was raised against an epitope, the epitope being a subcellular compartment polypeptide or a motor polypeptide as described herein). Non-limiting examples of which include polypeptides comprising one or more CDRs complementary to growth associated protein 43 (GAP43), doublecortin, or synaptosome-associated protein 25 (SNAP25), which can be utilized, for example, to target localization of RNAs of interest to the neuronal growth cone. 2.2.2 Motor Polypeptides

[0078] Motor polypeptides as described herein comprise one or more polypeptides that can utilize cellular energy (for example, the hydrolysis of ATP) to induce conformational changes that can generate mechanical forces or otherwise result in a net movement of the motor polypeptides. The skilled artisan would readily understand that such mechanical forces can then be used for physical translocation.

[0079] Without intending to be limiting, examples of motor polypeptides as described herein include: cytoskeletal motors such as a myosin polypeptide (for example, a myosin of myosin families I-XIV), a kinesin polypeptide (a polypeptide of the kinesin superfamily, such as KIF3B, KIF17, KIF13A, KIFC1, KIF5a, or KIF5b, for example), and a dynein polypeptide (for example, a cystoplasmic dynein comprising one or more DYNC1 or DYNC2 heavy chains, intermediate chains, light intermediate chains, or light chains, or an axonemal dynein comprising one or more dynein axonemal heavy chains (DNAHs), intermediate chains (DNAIs), light intermediate chains (DNALIs), or light chains (DNALs)); a polymerization motor polypeptide (such as a dynamin, or actin polymerization motors); a rotary motor polypeptide (such as an F0F1-ATP synthase); and a nucleic acid motor (such as a DNA polymerase, a DNA helicase, and a topoisomerase). 2.3. RNA Carrier Polypeptides

[0080] RNA carrier polypeptides as described herein are able to bind to one or more RNA in a cell. In some embodiments, the RNA carrier polypeptides preferentially or specifically bind to RNA having a specific sequence (for example, a “zipcode” sequence) or to a specific type of RNA (for example polyA RNA). In embodiments, RNA carrier proteins (encoded by RNA carrier polypeptides as described herein) according to the present disclosure may be sequence-specific, in that they recognize RNA having certain sequences. In embodiments, sequence-specific RNA carrier polypeptides can include, but are not limited to polypeptides that specifically bind to an RNA sequence in combination with a guide RNA. Binding of an RNA of interest to an RNA 25 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC carrier protein can allow for translocation of that RNA according to aspects of the present disclosure. 2.3.1. RNA carriers

[0081] RNA carrier polypeptides as described herein encode RNA carrier proteins that are capable of binding to and translocating RNA. Examples of RNA carrier proteins include trans- acting factor (TAF) proteins, protein-based RNA-binding domains, RNA-guided RNA-targeting CRISPR–Cas effectors, MS2 bacteriophage coat protein (MCP) which can bind (for example) RNA tagged with MS2 aptamers, and PP7 bacteriophage coat protein (PCP) which can bind (for example) RNA tagged with PP7 aptamers. Additional non-limiting examples of which include z- DNA brinding protein 1 (ZBP1), UPA1, RNA-binding protein 4 (RRM4) and polyadenylate- binding protein (PAB1), transport granule proteins such as fragile X messenger ribonucleoprotein 1 (FMRP) and splicing factor proline and glutamine rich (SFPQ), unk zing finger (UNK), PUF (Pumilio and FBF homology) domain (doi:10.1038 / nchembio.577), pentatricopeptide repeat proteins (PPRs) (doi:10.1146 / annurev-arplant-050213-040159), prokaryotic Argonaute (pAgo) system (doi:10.1073 / pnas.1717725115), CRISPR–Cas-inspired RNA-targeting systems (CIRTS) (doi:10.1016 / j.cell.2019.05.049), and type II (Cas9), type III (Csm / Cmr), or type VI (Cas13) CRISPR-associated protein (Cas) or dead Cas (dCas) proteins (doi:10.1038 / s41556-019-0454-7) (doi:10.3390 / ijms21031122). 2.3.2 Cas and dCas Polypeptides

[0082] Catalytically-inactive or “dead” Cas (dCas) RNA carriers (i.e., nuclease-dead) according to the present disclosure can comprise a type II (Cas9), type III (Csm / Cmr), or type VI (Cas13) CRISPR system dCas polypeptide comprising one or more mutations in a wild-type Cas (or other Cas having nuclease activity) that renders the Cas polypeptide catalytically-inactive (i.e., without nuclease activity, thereby becoming a dCas polypeptide). In embodiments, the dCas polypeptide is a dCas9 polypeptide, a dCas13 polypeptide (i.e., a dCas13a polypeptide, dCas13b polypeptide, dCas13c polypeptide, dPspCas13b polypeptide, or dCas13d polypeptide) or dCas7-11 polypeptide. See, for example, Burmistrz et al. Int. J. Mol. Sci. 2020, 21, 1122; doi:10.3390 / ijms21031122, including supplemental information, hereby incorporated by reference in its entirety, and O’Connell et al. Nature. 2014 December 11; 516(7530): 263–266, including supplemental information, hereby incorporated by reference in its entirety. 26 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0083] As used throughout, the term “Cas13 polypeptide” means a Cas13 protein or a fragment thereof present in any bacterial species that encodes a Type VI CRISPR / Cas13 system and that binds to RNA in combination with a suitable gRNA. See, for example, Abudayyeh et al., Science. 2016 August 5; 353(6299): aaf5573. doi:10.1126 / science.aaf5573, including supplemental information, hereby incorporated by reference in its entirety; Cox et al., Science 358, 1019–1027 (2017) including supplemental information, hereby incorporated by reference in its entirety; and Tang et al., Front. Cell Dev. Biol., 27 July 2021 Sec. Epigenomics and Epigenetics Volume–9 - 2021 | https: / / doi.org / 10.3389 / fcell.2021.677587. For example, the Cas13 protein or a fragment thereof with ssRNA targeting activity can be from Leptotrichia wadei, Leptotrichia shahii, Prevotella sp. P5-125 (PspCas13b), or Ruminococcus flavefaciens. Generally, Cas13 enzymes have two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) endoRNase domains that mediate precise RNA cleavage with a preference for targets with protospacer flanking sites (PFSs) observed biochemically and in bacteria.

[0084] As used throughout, a dCas13 polypeptide is a deactivated or nuclease-dead Cas13 (dCas13) that has been modified to inactivate Cas13 nuclease activity. Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. For example, and not to be limiting, H133A and H1058A mutations can be made in Cas13 HEPN domains from Prevotella sp. P5-125 (PspCas13b) to inactivate Cas13 nuclease activity (see, for example, Cox et al., Science 358, 1019–1027 (2017) including supplemental information, hereby incorporated by reference in its entirety, and International Patent Publication WO 2019 / 005884, also incorporated by reference in its entirety). Other modifications include removing all or a portion of the nuclease domain of Cas13 (for example, ǻ984-1090 H133A of Cas13b is from Prevotella sp. P5-125; see, for example, Programmable m(6)A modification of cellular RNAs with a Cas13-directed methyltransferase. Wilson C, Chen PJ, Miao Z, Liu DR. Nat Biotechnol. 2020 Jun 29. pii: 10.1038 / s41587-020-0572-6. doi: 10.1038 / s41587- 020-0572-6.10.1038 / s41587-020-0572-6 PubMed 32601430, incorporated by reference as if fully set forth herein, including supplementary information), such that the sequences exhibiting nuclease activity are absent from Cas13. Accordingly, a dCas13 may include polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The dCas13 retains the ability to target ssRNA even though the nuclease activity has been inactivated. Accordingly, dCas13 includes the polypeptide sequence or 27 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC sequences required for ssRNA targeting but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity. Additional examples of dCas13 polypeptides includes dRfxCas13d, dPguCas13b, dMisCas13b, dHgm4Cas13b, dHgm6Cas13b, dBba2Cas13b, and dPba3Cas13b (see, for example, Huang, Y., Gao, BQ., Meng, Q. et al. CRISPR-dCas13- tracing reveals transcriptional memory and limited mRNA export in developing zebrafish embryos. Genome Biol 24, 15 (2023). https: / / doi.org / 10.1186 / s13059-023-02848-6, the entirety of which is incorporated by reference as if fully set forth herein, including supplementary information).

[0085] In some examples, the dCas13 protein is a full-length Cas13 sequence from L. wadei, L. shahii, Prevotella sp. P5-125 (PspCas13b), or R. flavefaciens having one or more mutations in one or more HEPN domains and retaining the ssRNA targeting function. In other examples, the dCas13 protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to Cas13 polypeptide sequences with HEPN mutations and retains RNA binding function.

[0086] As used throughout, the term “Cas9 polypeptide” means a Cas9 protein or a fragment thereof present in any bacterial species that encodes a Type II CRISPR / Cas9 system. See, for example, Makarova et al. Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety. For example, the Cas9 protein or a fragment thereof with ssRNA targeting activity can be from Streptococcus pyogenes, Staphylococcus aureus, Campylobacter jejuni, or Neisseria meningitides. Generally, full-length Cas9 is an endonuclease comprising a recognition domain and two nuclease domains (HNH and RuvC, respectively) that creates double-stranded breaks in DNA sequences. In the amino acid sequence of Cas9, HNH is linearly continuous, whereas RuvC is separated into three regions, one left of the recognition domain, and the other two right of the recognition domain flanking the HNH domain. Cas9 from Streptococcus pyogenes is targeted to a genomic site in a cell, for example, by interacting with a guide RNA that hybridizes to a 20-nucleotide DNA sequence that immediately precedes an NGG motif recognized by Cas9. This results in a double-strand break in the genomic DNA of the cell.

[0087] As used throughout, a dCas9 polypeptide is a deactivated or nuclease-dead Cas9 (dCas9) that has been modified to inactivate Cas9 nuclease activity. Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. For example, and not to be limiting, D10A, C80S, C574S, and H840A mutations can be 28 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC made in Cas9 from Streptococcus pyogenes to inactivate Cas9 nuclease activity (see, for example, O’Connell et al. Nature. 2014 December 11; 516(7530): 263–266, including supplemental information, hereby incorporated by reference in its entirety). Other modifications include removing all or a portion of the nuclease domain of Cas9, such that the sequences exhibiting nuclease activity are absent from Cas9. Accordingly, a dCas9 may include polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The dCas9 retains the ability to target ssRNA even though the nuclease activity has been inactivated. Accordingly, dCas9 includes the polypeptide sequence or sequences required for ssRNA targeting but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity. It is understood that similar modifications can be made to inactivate nuclease activity in other site-directed nucleases, for example in Cpf1 or C2c2.

[0088] In some examples, the dCas9 protein is a full-length Cas9 sequence from S. pyogenes, S. aureus, C. jejuni, or N. meningitides lacking the polypeptide sequence of the RuvC nuclease domain and / or the HNH nuclease domain and retaining the ssRNA targeting function. In other examples, the dCas9 protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to Cas9 polypeptide sequences lacking the RuvC nuclease domain and / or the HNH nuclease domain and retains DNA binding function.

[0089] Additional examples of dCas proteins that can be utilized according to the present disclosure are provided in Table 1 below: Table I. Examples of dCas proteins that can be utilized for embodiments of CRISPR-TO systems described herein:29 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 2.3.3 Guide RNAs

[0090] A target-specific guide RNA comprises a nucleotide sequence that is complementary to a RNA target sequence, and thereby mediates binding of the Cas-gRNA complex by hybridization at the target site. RNA target sequences may be mRNA or other RNAs as described below.

[0091] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. For dCas systems as described herein that utilize crRNA and tracrRNA, the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules. 2.3.4 Translocated RNA

[0092] Exemplary types of RNA that can be translocated according to the present disclosure can include coding and non-coding RNAs, e.g., messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), micro-RNA (miRNA), small nuclear RNAs (snRNA; 150 nt), small nucleolar RNAs (snoRNA; 60-300 nt), piwi-interacting RNAs (piRNA; 24-30 nt), circular RNA (circRNA; 100-4,000 nt), enhancer RNA (eRNA; 50-2,000 nt), long noncoding RNAs (lncRNA).

[0093] Examples of mRNA transcripts that can be translocated include a glyceraldehyde-3- phosphate dehydrogenase (GAPDH, for example, UniProtKB / Swiss-Prot: P04406.3), a ȕ-actin (ACTB, for example, GenBank: AKI70837.1), a succinate dehydrogenase complex subunit C (SDHC, for example, GenBank: CAG33383.1), a succinate dehydrogenase complex subunit D (SDHD, for example, GenBank: CAG33213.1), a NADH:ubiquinone oxidoreductase core subunit S2 (NDUFS2, for example, GenBank: AAG13809.1), a post-synatic density protein 95 (Psd95, for example, UniProtKB / Swiss-Prot: P78352.3), a calcium / calmodulin dependent protein kinase II alpha (CamK2A or CamKIIa, (for example, GenBank: CCQ43527.1), a glutamate ionotropic receptor NMDA type subunit 2B (Grin2b, for example, NCBI Reference Sequence: NP_001400922.1), a C57L integrin-associated protein (CD47, for example, GenBank: CEJ95640.1), a B-cell lymphoma 2 (BCL-2, for example, NCBI Reference Sequence: NP_000624.2), a programmed death-ligand 1 (CD274 / PDL1, for example, GenBank: AAH69381.1), a tumor protein 53 (p53, for example, GenBank: BAC16799.1), an epidermal 30 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC growth factor receptor (EGFR, for example, GenBank: AAI18666.1), a g-protein coupled receptor (GPCR, or subunit thereof, for example, a metabotropic glutamate receptor subunit for example, GenBank: AAB49751.1), or a transferrin receptor (TFRC / CD71, for example, GenBank: AAH01188.1). In certain aspects, transcripts may be mammalian, for example human or rodent transcripts.^ 2.4. Dimerization Handles and Affinity Agents

[0094] In embodiments, instead of being translationally-fused to one another or directly covalently-linked, localization polypeptides and RNA carrier polypeptides can interact with each other through dimerization handles and affinity agents. For example, a localization polypeptide may be translationally fused with (or otherwise covalently or non-covalently associated with) a first dimerization handle, and the RNA carrier polypeptide may be translationally fused with (or otherwise covalently or non-covalently associated with) a second dimerization handle.

[0095] The first and second dimerization handles may then interact with each other and comprise complementary amino acid sequences (or other secondary or tertiary three-dimensional (3D) polypeptide structure beyond the primary amino acid sequence) capable of reversibly or irreversibly binding to one another. In this sense, the first and second dimerization handles may directly interact with one another to form a dimerization pair. The dimerization pair then can bring together a localization polypeptide and RNA carrier polypeptide so that RNA binds to the RNA carrier polypeptide and is then translocated in the cell by way of subcellular targeting through the localization polypeptide and the association of the RNA carrier polypeptide with the localization polypeptide. As should be apparent, dimerization pairs according to the present disclosure may comprise homodimeric systems (i.e., the first and the second dimerization handle are two polypeptides of the same identity, or substantially similar amino acid identity) or heterdimeric systems (i.e., the first and the second dimerization handle are different polypeptides).

[0096] The reader will appreciate that illustrations of polypeptides herein are schematic and not intended to be comprehensive. A variety of additional modifications (relative to wild-type PYL1 and ABI1 polypeptides, for example) are contemplated, including but not limited to combining elements separately described in different embodiments disclosed herein. Likewise, the skilled reader will be aware that modifications can be made to PYL1 and ABI1 polypeptide sequences involved in dimerization and assembly without loss of function. 2.4.1 Chemically-Induced Dimerization (CID) Handles 31 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0097] As described herein, the first and second dimerization handles may interact with one another indirectly. Such indirect interaction can a chemically-induced dimerization through an affinity agent, for example, a small molecule, polynucleotide, or polypeptide capable of binding both the first and the second dimerization handles, leading to the formation of a dimerization pair. In the instance of reversible dimerization through an affinity agent intermediate, the affinity agent can be administered to a call comprising a first and second dimerization handle, thereby forming a dimerization pair of the first and second dimerization handles. Such an inducible interaction, in embodiments, can be done in a dose-dependent manner so that dimerization increase as the intracellular concentration of an affinity agent increases. In the presence of an affinity agent, the dimerization pair then can bring together a localization polypeptide and RNA carrier polypeptide so that RNA binds to the RNA carrier polypeptide and is then translocated in the cell by way of subcellular targeting through the localization polypeptide and the association of the RNA carrier polypeptide with the localization polypeptide. This inducible interaction can then be reversed upon wash out of the affinity agent. The inducible interaction can also be reversed or terminated through the administration of other agents that compete for binding one the first dimerization handle or the second dimerization handle with the affinity agent.

[0098] Examples of dimerization handles and affinity agents as described herein include, but are not limited to: the pyrabactin resistance 1-like (PYL):ABI system that utilizes abscisic acid as an affinity agent (see, for example, U.S. Patent No. 10,221,426; U.S. Patent No. 10,934,559; US Patent No. 11,641,857; and International PCT Publication WO 2023 / 141591 A2 for additional information on aspects of the system, including PYL and ABI proteins (and modified variants thereof), as well as ABA and ABA analogs, which are incorporated by reference as if fully set forth herein); a dimerization pair of a first and a second FKBP12 polypeptide; a dimerization pair of a first and second FKBP polypeptide that utilizes a chemical inducer such as AP20187; cyclophilin-calcineurin based systems; FKBP-Calcineurin systems; FKBP-CyPFas systems; FKBP-FRB domain of mTOR based systems; Gal-GID1 based systems; SNAPTag-HaloTag based systems; eDHFR-HaloTag based sytems; Bclxl-Fab (AZ1) based systems; GyrB-GyrB based systems; and cyclophilin-based systems.

[0099] In any of the aforementioned examples, one component can be the first dimerization handle while the other acts as the second dimerization handle (i.e., PYL can be the first dimerization handle while ABI can be the second dimerization handle, and vice versa, and the 32 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC affinity agent can induce dimerization regardless of which component is the first dimerization handle or the second dimerization handle). Additional examples of chemically-induced dimerization pairs and systems can be found, for example, in: Voß et al. Current Opinion in Chemical Biology 2015, 28:194–201; Lamb et al. STAR Protoc 2022 Apr 14;3(2):101323; and Dang Front. Chem., 08 February 2022 Sec. Supramolecular Chemistry Volume 10 - 2022 | https: / / doi.org / 10.3389 / fchem.2022.829312, the entireties of all of which are incorporated by reference as if fully set forth herein. 2.4.2 Optically-Induced Dimerization Handles

[0100] Optically-induced dimerization systems are also contemplated by the present disclosure. Whereas chemically induced dimerization (CID) systems are based on small molecules interacting with two identical proteins (homodimerization) or two different proteins (heterodimerization). Optogenetic dimerization systems employ photosensitive proteins that undergo a conformational change upon illumination, and consequently, induce protein interaction. Chemo^optogenetic dimerization systems can use photoactivatable and / or ^cleavable small^molecule dimerizers, so that proximity can be induced and / or disrupted by light.

[0101] Examples of optically-inducible dimerization systems include UVR8-COP1; UVR8- UVR8 homodimer systems; FKF1-G1; TULIPs; LOVpep-ePDZ; iLID; LOVSsrA-SsrB; Light on; VVD-VVD; and pMag-nMag (VVD variants). As with the CID systems described above, the skilled artisan would readily understand that in any of the aforementioned examples, one component can be the first dimerization handle while the other acts as the second dimerization handle, and vice-versa. Additional information on optically-inducible dimerization systems can be found, for example, in Klewer et al., Chemistry. 2019 Sep 25; 25(54): 12452–12463, which is incorporated by reference in its entirety as if fully set forth herein (including any supplemental information). 2.5 Cells

[0102] Cells as described herein can be a cell of the animal or plant kingdom. In embodiments, cells as described herein may be a mammalian cell, for example a human, mouse, or rat cell. In embodiments, the cell can be a plant cell. In embodiments, the cell is not a plant cell. 2.5.1 Mammalian Cells

[0103] Mammalian cells as described herein may be derived from immortalized cell lines or other primary cell lines derived from a subject (for example, a human subject). Mammalian cells, 33 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC for example, may be derived from any cellular germ layer, for example, mesoderm, endoderm, or ectoderm, or from any organ of the body (for example, liver hepatocytes or kidney cells, such as human embryonic kidney). In embodiments, mammalian cells may be placental or embryonic. Cells may be stem cells (for example, pluripotent cells such as human embryonic cells, or multipotent cells such as hematopoietic stem cells (HSCs) or bone-marrow derived mesenchymal stem cells (BM-MSCs)); bone cells (for example, osteoblasts or osteoclasts); blood cells (for example, white blood cells); muscle cells (also known as myocytes); sperm cells; a female egg; skin cells; endothelial cells; epithelial cells; fat cells; cells of the central or peripheral nervous system (for example, neurons, glia, and pericytes); or cells from an organ in the body, such as kidney or liver.

[0104] Without intending to be limiting, in embodiments, the cell can be: a neuron; a glial cell (i.e., an astrocyte, oligodendrocyte, or Schwann cell); a pericyte; a fibroblast; an intestinal epithelial cell; a mesenchymal cell; a T cell; a cancer cell; a stem cell; a chondrocyte; an osteoblast; an osteoclast; an osteocyte; a HSC; a BM-MSC; an induced pluripotent stem cells; an embryonic stem cells; a granulocyte; an agranulocyte; a skeletal, cardiac, or smooth muscle myocyte; or an adipocyte (i.e., a white or brown adipocyte). 2.5.2 Plant Cells

[0105] In embodiments where the cell is a plant cell, a plant cell can be a parenchymal, collenchymal, sclerenchymal, xylem, phloem, meristematic, or epidermal plant cell. Generally, plant cells according to the present disclosure may include eukaryotic cells with large central vacuoles, cell walls containing cellulose, and plastids such as chloroplasts and chromoplasts. Additionally, plant cells: may be non-motile; may make their own food (i.e., are autotrophic); may reproduce asexually by vegetative propagation or sexually; may contain an outer cell wall and a large central vacuole; may contain photosynthetic pigments (i.e., a chlorophyll) that can be present in the plastids; and may have different organelles for anchorage, reproduction, support and photosynthesis. 2.6 Compositions

[0106] Compositions according to the present disclosure may comprise any one or more aspects of the present disclosure discussed herein. Compositions, for example, may comprise any one or more localization polypeptides, motor polypeptides, RNA carrier polypeptides, dimerization handles, or cells as described herein. 34 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0107] Compositions may be provided in any number of forms as known in the art. Without intending to be limiting, compositions may be provided aliquots frozen in liquid nitrogen; lyophilized preparations; liquid compositions comprising a biologically- or pharmaceutically- acceptable medium (for example, phosphate-buffered saline or other buffered or non-buffered cell growth medium comprising combinations of sugars (such as glucose), cytokines, growth factors, amino acids, vitamins, inorganic salts, and serum). 2.6.1 Polypeptides

[0108] As would be understood from the present disclosure, described herein are compositions comprising one or more polypeptides according to the present disclosure. Compositions as described herein may comprise one or more polypeptides from any of sections 2.1-2.4 above. 2.6.2 Peptide Linkers

[0109] In embodiments of fusion proteins according to the present disclosure, linker sequences may be present that join two or more coding polypeptides (a coding polypeptide being a polypeptide that encodes a protein according to the present disclosure). In certain aspects, linkers may be cleavable or non-cleavable (based on the nature of their cleavage site). Cleavable linkers can be enzyme-sensitive, pH-sensitive, and glutathione sensitive whereas non-cleavable linkers can include very stable amide, carbon or ether chains (see, for example, Fu C, Yu L, Miao Y, Liu X, Yu Z, Wei M. Peptide–drug conjugates (PDCs): a novel trend of research and development on targeted therapy, hype or hope? Acta Pharm Sin B. 2022.)

[0110] Non-limiting examples of linker sequences according to the present disclosure can be found, for example, in Table 2 below. Additional examples can include polyethylene glycols (PEGs), Table 2: Embodiments of Linker Sequences According to the Present Disclosure2.6.3 Polynucleotides 35 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC

[0111] Also provided herein are polynucleotides (e.g., RNA or DNA) that encode any of the polypeptides described, for example those as described in sections 2.1-2.4 above. Optionally, expression cassettes comprising a promoter operable linked to the coding sequences, which may be in a vector or other viral vector are contemplated, in addition to other regulatory sequences that may be required for translation of the polynucleotide from the vector.

[0112] Additionally described herein are vectors and compositions comprising one or more vectors. Vectors as described herein may comprise any one or more polynucleotides encoding any one or more polypeptides according to the present disclosure. Vectors as described herein may be non-viral plasmids that can be delivered to a cell through non-viral delivery methods, for example, transfection, electroporation, and nucleofection. Vectors as described herein may also be viral vectors, for example, lenti, adeno-associated, or baculoviral vectors that can be packaged into functional viruses that can transduce cells as described herein.

[0113] Compositions as described herein can include single, dual, or triple vector systems, for example, wherein polynucleotides are present in one vector, two vectors, three vectors, and the like. Compositions as described herein can also comprise a physiologically suitable buffer, for example phosphate-buffered saline, that can be utilized according to the present disclosure. 2.7 Methods of Use

[0114] Described herein are methods of use of polypeptides, polynucleotides, and cells as described herein. 2.7.1 Advantages of Methods over Existing Methods

[0115] Aspects of embodiments of methods described herein can provide at least the following advantages over existing methods: 1. applications of combinations of polypeptides as described herein can manipulate RNA localization by using RNA-guided catalytically-dead enzymes (for example, dCas13) to bind target RNAs, which does not require the prior knowledge of localization elements; 2. applications of combinations of polypeptides as described herein provide the added convenience of perturbing the localization of different endogenous RNAs by just changing the corresponding gRNAs used, while the other systems (such as the PULR system) needs to reprogram PUF domains for each target RNAs; 3. there is no genomic manipulation on the sequences of the target RNA using combinations of polypeptides as described herein, thereby minimizing the potential influence on key properties (translation, stability, etc.) of the target RNA; 4. combinations of polypeptides as described herein can be applied for high-throughput screening 36 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC to manipulate the subcellular localization of hundreds to thousands of RNAs by preparing gRNA libraries, which cannot be achieved by any of the current strategies; 5. combinations of polypeptides as described herein can be utilized in an inducible system, which can manipulate the subcellular localization of target RNA with higher temporal precision (i.e., be turned “on” and “off”); and 6. combinations of polypeptides as described herein can be applied to manipulate RNA localization in situ and in vivo using animal models, which is a major bottleneck to link RNA spatial information to physiology and disease. 2.7.2 Methods of RNA Localization

[0116] Provided herein are methods of localizing RNA to subcellular (i.e., intracellular or specific intracellular) locations. In some embodiments, polypeptides and / or polynucleotides as described herein can be introduced into a cell, and RNA can be localized to a subcellular location by binding to an RNA carrier polypeptide, which can then be translocated upon binding of the localization polypeptide to its subcellular target. This can happen without additional steps in embodiments utilizing translationally-fused localization and RNA carrier polypeptides. Other configurations of localization polypeptides and RNA carrier polypeptides may also require the steps of dimerization of the dimerization handles (either chemically or optically, for example), or the use of a guide RNA to assist in binding the RNA of interest to the RNA carrier polypeptide.

[0117] In some embodiments, the methods comprising providing one or more cells comprising the RNA carrier polypeptide and the localization polypeptide and allowing the RNA carrier polypeptide to bind to the target RNA(s) and localizing the RNA bound to the RNA carrier polypeptide to the subcellular location with the localization polypeptide. A gRNA can be provided to the one or more cells (either by direct injection, transfection, lipofection, or other similar technique, or expression from a vector that is introduced to the cell according to methods known in the art) that is complementary to (or otherwise has a sequence designed to allow for specific binding) an RNA of interest. The RNA of interest can be an endogenous or exogenous RNA that one desires to move to a specific subcellular location in a cell.

[0118] For embodiments with translationally-fused (or otherwise covalently-linked) localization polypeptides and RNA carrier polypeptides, such localizing can be done by localization and RNA carrier polypeptides binding to their respective targets. In embodiments with dimerization handles, the localization and RNA carrier polypeptides can be dimerized in the presence of a chemical inducer (i.e., an affinity agent) or an optical inducer (i.e., a laser comprising or consisting 37 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC essentially of light of a given wavelength that can introduce a conformational change in a dimerization handle as described herein). Following dimerization, the localization and RNA carrier polypeptides can bind to an RNA of interest (i.e., an endogenous mRNA or other exogenous RNA) and then be drawn to the targeted subcellular location, thereby bringing the RNA of interest along. In embodiments utilizing catalytically-dead RNA-guided enzymes (such as catalytically- dead nucleases, i.e., dCas polypeptides), a guide RNA can be introduced into a cell along with the localization and RNA carrier polypeptides that can assist in specifically binding an RNA of interest to the RNA carrier polypeptides, which can then be localized to a subcellular location based on binding of the localization polypeptide to its target. 2.7.3 Intracellular Polypeptide Introduction

[0119] Without intending to be limiting, polypeptides as described herein can be injected / extrapolated as proteins by microscale or nanoscale injection, bulk and localized electroporation, use of cationic lipids and polymers and other cationic amphiphilic-based delivery reagents, coadministration / coexpression or fusion with cell-penetrating peptides or toxins, lipid bilayer deformation, peptide resurfacing, coadministration / coexpression or fusion with endosomal and lysosomal signaling sequences, and the like (see, for example, Virginia J. Bruce, Brian R. McNaughton, Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells, Cell Chemical Biology, Volume 24, Issue 8, 2017, pp. 924-934, Weill CO, Biri S, Adib A, Erbacher P. A practical approach for intracellular protein delivery. Cytotechnology. 2008 Jan;56(1):41-8. doi: 10.1007 / s10616-007-9102-3. Epub 2007 Oct 16. and Chau, C, Actis, P, and Hewitt, E (2020) Methods for protein delivery into cells: from current approaches to future perspectives. Biochemical Society Transactions, 48 (2). pp. 357-365, which are incorporated by reference as if fully set forth herein). Introduction of polypeptides described herein can be introduced into cells in vitro, ex vivo or in vivo. Ex vivo refers to introduction of the polypeptides outside the body of a subject and subsequent introduction of the cells into the subject. The cells can be autologous or allogeneic. 2.7.4 Intracellular Polynucleotide Introduction

[0120] Polypeptides as described herein may also be introduced into a cell by translation from a vector encoding the polypeptide, which can be introduced into a cell. The vectors comprising polynucleotides as described herein can be introduced into cells in a manner suitable for subsequent transient or stable expression of polynucleotides as described herein. The method of 38 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC introduction can be dictated by the targeted cell type or the vector size (more specifically the size of the transgene in the vector), discussed below. Exemplary methods include CaPO4precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral vector introduction which can include, e.g. lentiviral, AAV, adenoviral or other viralinfection (i.e., transduction), dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection. Introduction of polynucleotides or vectors described herein can be introduced into cells in vitro, ex vivo or in vivo. Ex vivo refers to introduction of the polynucleotides or vectors outside the body of a subject and subsequent introduction of the cells into the subject. The cells can be autologous or allogeneic. 2.7.5 Embodiments of Applications of the Present Technology

[0121] The reader would readily understand that aspects of the present disclosure (in particular applications of the technology as described above) are useful in at least the following non-limiting areas / prophetic applications: basic research (for example, to decipher the fundamental mechanisms and functional roles of subcellular RNA localization by manipulating the subcellular localization of endogenous RNA followed by analysis of the corresponding functional influence; disease research (for example to investigate the pathological mechanism of diseases (ALS, FXS, SMA, etc.) related with mislocalized RNAs and screen out the candidate mRNA for therapeutics); gene therapy (for example to regulate the protein expression level of target genes without any genetic manipulation by targeting transcriptional repressors); regulation of a level of protein expression of a gene of interest at specific subcellular compartments (for example, translocating the target mRNA to the growth cone of neurons and increase its protein level at growth cone by local translation of the mRNA; RNA medicine (i.e., RNA therapeutics by correcting mistargeted RNA).

[0122] Additional examples of methods of the present disclosure can be seen in the Examples section below. 2.8 Kits^

[0123] Compositions as described herein can be part of a kit. Kits comprising one or more vectors comprising nucleic acids encoding polypeptides as described herein. Kits may comprise compositions as described herein (for example, vectors, nucleic acids, and polypeptides) in a frozen aqueous solution, or dry lyophilized preparation, for example. Such preparations can be reconstituted by an end user by methods as known in the art. Such a kit may further comprise a carrier means being compartmentalized to receive in close confinement one or more container 39 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC means such as vials, tubes, and the like. The kit may also have containers containing buffer(s) and instructions for use. Kits may additionally include aspects of the present disclosure that help enable RNA binding, such as gRNA, or affinity agents that aid in dimerization of dimerization handles as discussed herein (such as abscisic acid (ABA), for example). Kits may also include instructions for using the components of the kit. EXAMPLES

[0124] The following examples are offered to illustrate, but not to limit, aspects of the present disclosure. Example 1 – Development of the technology for reporter RNAs.

[0125] To develop CRISPR-TO (FIG. 1), a dCas13-ABI construct was optimized for high binding affinity with the target RNA and good solubility in cells by testing different protein fusion orders (dPspCas13b-linker-BFP-ABI and ABI-dPspCas13b-linker-BFP), linkers (Linker 1, Linker 2, and Linker 3, see Table 2), and fluorescent proteins (BFP and EGFP) between dCas13 and ABI, and the construct of dPspCas13b-Linker2-EGFP-ABI (referred as dCas13-ABI) was finally selected. Since how many binding sites of dCas13-ABI are needed to reposition a target mRNA was not clear at the beginning, a reporter mRNA (Yang et al., 2019) with 24xGCN4 repeats in the 3’UTR to establish CRISPR-TO in HeLa cells was tried first. dCas13-ABI was targeted to the 24xGCN4 repeats region which can provide 24 binding sites for dCas13-ABI by only expressing one guide RNA (gRNA). PYL1 was then fused to the C-terminal 31 amino acids of mitochondrial anti-viral signaling protein (named as MAVS*-PYL1) to localize it to the outer mitochondria membrane (OMM). HeLa cells were transiently transfected with the plasmids expressing three CRISPR-TO components (dCas13-ABI, MAVS*-PYL1, and gRNA) and the reporter mRNA followed by ABA treatment and analyzed the localization of reporter mRNA by RNA FISH imaging. As expected, the reporter mRNA was efficiently repositioned to OMM which is both targeting gRNA-dependent and chemical inducible (FIG. 2A and 2B). To investigate how many binding sites of dCas13-ABI are required for efficient repositioning of a target mRNA, a series of reporter mRNAs were tested with different numbers of GCN4 repeats (GCN4x1, GCN4x2, GCN4x3, GCN4x7, and GCN4x24). Efficient repositioning was observed from GCN4x2 reporter mRNA, suggesting that only two binding sites of dCas13-ABI are already enough for CRISPR- TO to manipulate the localization of the target mRNA efficiently (FIG. 2C). 40 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC Example 2 – Development of the technology for endogenous RNAs.

[0126] Next, it was tested whether CRISPR-TO can be applied to manipulate the localization of endogenous mRNAs. GAPDH mRNA was chosen as a candidate endogenous mRNA to be tested here. Three gRNAs (gG1, gG2, and gG3) targeting the 3’UTR region of GAPDH mRNA were designed using the Ācas13design” tool Wessels et al., 2020). The 3’UTR region of mRNA was chosen to design gRNAs considering that the binding of dCas13-gRNA complex on the 5’UTR region may block translation initiation and the dCas13-gRNA complex bound on the CDS region may be removed by the translating ribosomes. To increase the binding affinity between gRNA and the target RNA, the length of spacer was increased to 30 nt.

[0127] HeLa cells were transiently transfected with the plasmids expressing two CRISPR-TO protein components (dCas13-ABI and MAVS*-PYL1) and the mixture of three gRNAs targeting GAPDH mRNA (gG123) followed by ABA treatment and analyzed the localization of GAPDH mRNA by RNA FISH imaging. Like the reporter mRNA, the endogenous GAPDH mRNAs were efficiently repositioned to OMM which is both targeting gRNA-dependent and chemical inducible (FIG.3A-3B). Different combinations of the three gRNAs of GAPDH were also tested and found that two gRNAs are already enough for the efficient repositioning of GAPDH mRNAs (FIG.3C).

[0128] Furthermore, it was tested to see if CRISPR-TO can be broadly applied to various endogenous mRNAs by manipulating four more endogenous mRNAs with different expression levels (ACTB, SDHC, SDHD, and NDUFS2). It was found that CRISPR-TO can reposition all these mRNAs to OMM efficiently, indicating its broad applicability to various endogenous mRNAs. CRISPR-TO was also applied to reposition endogenous mRNA to OMM in different cell lines (HEK293T and Neuro-2a cell lines) and it also works efficiently.

[0129] CRISPR-TO can be modified to reposition mRNA to different subcellular compartments by changing the protein fused with PYL1. Besides OMM, we tested six more subcellular compartments which all work for CRISPR-TO (FIG.4). For localizing mRNA to p-bodies which were originally thought to be sites of mRNA decay, PYL1 was fused with DDX6 which is a marker protein of p-body. For localizing mRNA to stress granules, PYL1 was fused with G3BP1. The recruitment of mRNA to OMM, p-body, and stress granules by CRISPR-TO is mediated by the passive diffusion of dCas13-ABI-bound mRNA which is immobilized by ABA ligand after approaching the cognate heterodimer partner PYL1. Another widely applied mechanism for RNA 41 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC localization in cells is to actively transport mRNA along microtubules using motor proteins. We imitated this mechanism by hijacking motor proteins to transport target mRNAs toward the plus or minus ends of microtubules by fusing PYL1 with a truncated motor protein KIF5b(aa1-555) (named as KIF5b*) or a truncated motor protein KIFC1(aa125-673) (named as KIFC1*), respectively. Besides these cytosolic compartments, we also tested two nuclear bodies telomere and nuclear stress bodies by fusing PYL1 with TRF1 which is a marker protein of telomere or HSF1 which is a marker protein of nuclear stress bodies, respectively (FIG. 5). In conclusion, CRISPR-TO can be applied to manipulate the localization of various endogenous mRNAs to different subcellular compartments in diverse cell lines. Example 3 – Real-time monitoring of RNA localization manipulation

[0130] CRISPR-TO is designed as a chemical-inducible system and we plan to characterize its dynamic properties. We first tested if the repositioning efficiency of target mRNAs can be adjusted by changing the concentration of ABA by applying CRISPR-TO to reposition GAPDH mRNA to OMM under different concentrations of ABA. We found that the percentage of GAPDH mRNA localized on OMM increased gradually with the increase of ABA concentration and treating cells with 5 μM ABA for 4 hours already triggered the significant enrichment of GAPDH mRNA on OMM (FIG.6A).

[0131] We next treated cells with 250 μM ABA for different periods (10 min, 20 min, 30 min, 1 h, 2 h, 4 h, and 24 h) and quantified the percentage of dCas13-ABI and GAPDH mRNA localized on OMM after CRISPR-TO perturbation. We found that dCas13-ABI and GAPDH mRNA were gradually enriched on OMM over time, but dCas13-ABI was recruited to OMM faster than GAPDH mRNA. dCas13-ABI began to be significantly enriched on OMM after 10-min treatment of ABA, while GAPDH mRNA began to be significantly enriched on OMM after 1-hour treatment of ABA (FIG.6B). One possible explanation is that there are two populations of dCas13-ABI with different diffusive velocities. Free dCas13-ABI proteins which do not bind on mRNA diffuse more quickly, leading to faster attachment on OMM, while dCas13-ABI bound on mRNA diffuse more slowly as RNP complexes, leading to slower attachment on OMM. To test if the changing of mRNA localization by CRISPR-TO is reversible, we first treated cells with 250 μM ABA for 4 hours and then removed ABA for different periods (10 min, 20 min, 30 min, 1 h, and 2 h). We found that dCas13-ABI and GAPDH mRNA were released gradually and with similar speed from 42 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC OMM after removing ABA, and were mostly released 1 hour after removing ABA (FIG. 6C). These results demonstrate that the CRISPR-TO system is chemically inducible and reversible.

[0132] Next, we investigated the real-time dynamics of mRNA localization manipulated by CRISPR-TO. For live-cell tracking of mRNA, we applied the U2OS-2-6-3 cell line which expresses a Dox-inducible CFP reporter mRNA fused with 24xMS2 repeats in its 3’UTR (named as MS2-mRNA) (Janicki SM, Tsukamoto T, Salghetti SE, Tansey WP, Sachidanandam R, Prasanth KV, Ried T, Shav-Tal Y, Bertrand E, Singer RH, Spector DL. From silencing to gene expression: real-time analysis in single cells. Cell.2004 Mar 5;116(5):683-98. doi: 10.1016 / s0092- 8674(04)00171-0. PMID: 15006351; PMCID: PMC4942132.). We first transduced U2OS-2-6-3 cells with lentivirus expressing rtTA and stdMCP-tdTomato which enables the live-cell tracking of the MS2-mRNA by binding to the 24xMS2 repeats and got the stable cells named U2OS-2-6- 3_MCP. Then we delivered the CRISPR-TO components (dCas13-ABI, MAVS*-PYL1, and one gRNA targeting MS2) into U2OS-2-6-3_MCP cells by electroporating plasmids (FIG. 6D). We tracked the real-time dynamics of MS2-mRNA after adding ABA and found that MS2-mRNA can be detected enriched on OMM only after 15-min treatment of ABA. We also tracked the real-time release of MS2-mRNA from OMM after removing ABA and found that MS2-mRNAs were mostly released from OMM after removing ABA for about 50 min which is consistent with what we observed for GAPDH mRNA.

[0133] To track the dynamics of MS2-mRNA transported by CRISPR-TO using motor proteins, we delivered the CRISPR-TO components (dCas13-ABI, KIFC1*-PYL1, and one gRNA targeting MS2) into U2OS-2-6-3_MCP cells by electroporating plasmids. After adding ABA, MS2-mRNAs were gradually enriched near the centromere. We tracked the trajectories of 60 mRNA particles with 3 s per frame and ranked them with their explored distance from smallest to largest. We can clearly see two populations of mRNA: type I shows confined, sub-diffusive motion and type II shows super-diffusive motion which might be mRNA under transportation. We calculated the Mean Square Displacement (MSD) of the first 20 and last 20 trajectories. The first 20 trajectories could belong to type I RNA with its MSD curving down at larger lag time and the last 20 trajectories could belong to type II RNA with its MSD curving up at larger lag time. We then calculated the distance of mRNA particle to the centromere as a function of time for several representative trajectories of each type. For trajectories of type II, the distance decreases over time, indicating that the mRNA is being transported to the centromere by CRISPR-TO components. For 43 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC trajectories of type I, the distance to ROI fluctuates, indicating the mRNA is not manipulated by CRISPR-TO under non-directed motion (FIG.6E). Example 4 – Applying CRISPR-TO in primary neurons to manipulate mRNA localization in the neuronal projections

[0134] One advantage of CRISPR-TO is that it does not need genomic engineering and can be applied directly in primary cells. To demonstrate this property, we applied CRISPR-TO in primary neurons to perturb the axonal localization of endogenous mRNAs by using the truncated motor protein Kif5a(aa1-559) (named as Kif5a*) which moves toward the plus end of microtubules which is also the distal end of neuronal projections (FIG. 7A). ACTB mRNA was chosen as a candidate to be tested here. We delivered the CRISPR-TO components (dCas13-ABI, Kif5a*- PYL1, and six gRNAs targeting ACTB mRNA) together with a fluorescent membrane protein (CAAX-Crimson) which shows the shape of neurons into mouse hippocampal neurons by plasmid transfection. We tiled multiple images together to show the full view of one neuron. Without ABA treatment (i.e., gT+DMSO group), dCas13-ABI proteins are mainly distributed in the cytoplasm of somas and there are almost no ACTB mRNAs in the terminals of neuronal projections. After treating neurons with ABA for 24 hours (i.e., gT+ABA group), dCas13-ABI proteins together with ACTB mRNAs were transported toward the plus end of microtubules and enriched in the terminals of neuronal projections (FIG. 7B). In contrast, only dCas13-ABI proteins, instead of ACTB mRNAs, were enriched in the terminals of neuronal projections after changing targeting gRNAs (gT) to gNT (i.e., gNT+ABA group) (FIG. 7C). In the gT+ABA group, ACTB mRNAs were enriched in multiple projection terminals of one neuron with some terminals close to the soma (FIG. 7B, inset 1, 328 μm) and some terminals much far away from the soma (FIG. 7B, inset 3, 788 μm).

[0135] We quantified the fluorescence intensity of ACTB mRNA, dCas13-ABI, and Kif5a*- PYL1 in the projection terminals. As expected, the amount of Kif5a*-PYL1 in the projection terminals is similar among the three groups. After adding ABA, dCas13-ABI proteins were transported to the projection terminals and accumulated there, but only in the gT+ABA group were there more ACTB mRNAs enriched in the projection terminals. Interestingly, there are more dCas13-ABI proteins located in the projection terminals in the gT+ABA group than in the gNT+ABA group. One possible explanation is that ACTB mRNA brought more dCas13-ABI to the projection terminals. We next quantified the number of ACTB mRNA particles located to the 44 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC projection terminals by CRISPR-TO based on the median fluorescence intensity of one ACTB mRNA particle and found that there are on median four ACTB mRNA particles enriched in one projection terminal in the gT+ABA group with some projection terminals having as many as 79 ACTB mRNAs. In the gT+ABA group, ACTB mRNA particles were often observed inside the neuronal projections. We quantified the number of ACTB mRNA particles per μm in neuronal projections and found that there are indeed more ACTB mRNAs inside neuronal projections in the gT+ABA group than the other two control groups. These mRNAs may be mRNAs being transported toward the distal end of microtubules by Kif5a*-PYL1. We quantified the distance between two adjacent ACTB mRNA particles in the neuronal projections of the gT+ABA group and found that the distance is widely distributed but mostly around 2 μm. In conclusion, CRISPR- TO can promote the localization of endogenous mRNAs in neuronal projections and projection terminals. Example 5 – Repositioned mRNAs by CRISPR-TO can be translated locally in the projection terminals of primary neurons

[0136] To analyze if the mRNA repositioned to the projection terminals of neurons can be translated into proteins locally, we designed a reporter mRNA (named NLS-HaloTag-GCN4x24) expressing a nuclear localized HaloTag protein with 24 GCN4 repeats in its 3’UTR (FIG. 8A). Without CRISPR-TO manipulation, reporter mRNAs are localized in the soma and the translated NLS-HaloTag proteins are mostly imported into the nucleus. After transporting reporter mRNAs to the projection terminals by CRISPR-TO, if the repositioned reporter mRNAs can be translated locally, fluorescent signals of HaloTag proteins should be detected there (FIG.8B). We delivered the CRISPR-TO components (dCas13-ABI, Kif5a*-PYL1, and gGCN4) together with CAAX- Crimson and NLS-HaloTag-GCN4x24 reporter mRNA into mouse hippocampal neurons by plasmid transfection. Before adding ABA, HaloTag proteins are mainly located in the nucleus and not detectable in the projection terminals. After adding ABA for 24 hours, there are HaloTag signals in the projection terminals of some neurons. As a control, there is no HaloTag signal in the projection terminals of the gNT+ABA group (FIG. 8C). We quantified the ratio of HaloTag protein signals in the projection terminal and soma and found that the ratio of gGCN4+ABA group is significantly higher than that of the two control groups, suggesting that repositioned mRNAs by CRISPR-TO can be translated locally in the projection terminals of mouse hippocampal neurons (FIG. 8D). 45 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC Example 6 – The axonal localization of ACTB mRNA regulates the growth of axons

[0137] To analyze if the perturbation of ACTB mRNA localization by CRISPR-TO influences the growth of neuronal axons, we applied CRISPR-TO in Neuro-2a cells which have been widely used to study axonal growth and neuronal differentiation (Tremblay RG, Sikorska M, Sandhu JK, Lanthier P, Ribecco-Lutkiewicz M, Bani-Yaghoub M. Differentiation of mouse Neuro 2A cells into dopamine neurons. J Neurosci Methods. 2010 Jan 30;186(1):60-7. doi: 10.1016 / j.jneumeth.2009.11.004. Epub 2009 Nov 10. PMID: 19903493.). We first delivered the CRISPR-TO components (dCas13-ABI, Kif5a*-PYL1, and 3 gRNAs targeting ACTB mRNA) together with CAAX-Crimson into Neuro-2a cells by plasmid transfection. One day after transfection, we treated Neuro-2a cells with ABA which initiates CRISPR-TO perturbation and Retinoic acid which induces the neuronal differentiation and neurite growth of Neuro-2a cells for two days. We analyzed the localization of ACTB mRNA and the length of axons two days later. Compared with gNT+ABA group which should not influence the localization of ACTB mRNAs theoretically, the localization of ACTB mRNAs in axonal tips and axons was significantly increased in the gT+ABA group, but significantly decreased in the gT+DMSO group possibly due to the trapping of ACTB mRNA by dCas13-ABI proteins mostly located in the cytoplasm of the soma (FIG. 9A-9C). To explore the influence of ACTB mRNA localization on the growth of axons, we first compared the axon length of Neuro-2a cells treated with ABA or DMSO, and the axon length of Neuro-2a cells in gNT+ABA or gNT+DMSO group to demonstrate that ABA does not influence the growth of axons in Neuro-2a cells. Next, we quantified the axon length of Neuro- 2a cells in gT+ABA and gT+DMSO group and found that the axon length of gT+ABA group is significantly shorter than that of gT+DMSO group. In addition, compared with Neuro-2a cells treated with DMSO, the axon length of gT+ABA group is shorter and the axon length of gT+DMSO group is longer (FIG. 9D-9E). These results indicate that promoting the axonal localization of ACTB mRNA blocks the growth of axons while blocking the axonal localization of ACTB mRNA promotes the growth of axons. Example 7 – Functionality of CRISPR-TO for manipulating RNA for mitochondria localization.

[0138] CRISPR-TO was applied to study the functional influence of OMM-localized mRNAs. There are many mRNAs identified to be localized on OMM (Fazal et al., 2019; Wang et al., 2019). However, the functions of such OMM-localization of mRNAs are not fully understood yet. There 46 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC are some hypotheses that OMM-localization of mRNA may enhance its local translation and the import of translated proteins into mitochondria, but there is very few evidence supporting these hypotheses. To test it, CRISPR-TO was applied to promote the OMM-localization of three reporter mRNAs expressing mRuby3, MTS-mRuby3, and MTS-mRuby3-DHFR respectively (FIG.10A). MTS is a canonical mitochondrial localization signal which can import proteins into mitochondria. DHFR (dihydrofolate reductase) is a degron which triggers the fast degradation of the fused protein. After recruiting the two reporter mRNAs to OMM using MAVS-PYL1 by CRISPR-TO, their protein level was analyzed using flow cytometry. It was found that only the protein level of MTS-mRuby3-DHFR reporter mRNA was significantly increased after recruiting its mRNAs to OMM, while the protein level of mRuby3 and MTS-mRuby3 was not significantly increased after recruiting its mRNAs to OMM (FIG. 10B). Other proteins (TOM20, TOM22, and TOM70) localized on OMM were also tested, and it was found that the protein level of MTS-mRuby3- DHFR reporter mRNA was also significantly increased using TOM22-PYL1 and TOM70-PYL1, but not TOM20-PYL1 (FIG. 10C). This indicates that OMM-localization of mRNA does not enhance its local translation significantly but promotes the import of translated proteins into mitochondria (FIG.10D). Example 8 – High throughput manipulation of subcellular mRNA localization in neurons.

[0139] One big advantage of CRISPR-TO is the feasibility of high-throughput screening, since gRNA libraries can be easily prepared to target thousands of genes. This large-scale screening application cannot be achieved by any of the current approaches. According to this prophetic example, a list of at least 1,000 mRNAs determined to be localized in neuronal axons can be generated. A high-content, phenotype imaging-based CRISPR screening in mouse primary cortical neurons can then be undertaken to explore the functional relevance of axonal localization for these 1,000 mRNAs by blocking the native axonal localization of each mRNA using CRISPR-TO and analyzing the functional influence by high-content imaging. Specifically, a library of gRNA-arrays targeting each of the 1,000 mRNAs can be constructed. Mouse primary cortical neurons can be seeded in 96-well culture plates and transfect the cells with plasmids or mRNAs expressing dCas13-ABI, KIF5C1*(aa125-673)-PYL1, and one gRNA-array targeting one candidate mRNA. ABA can then be added to block the axonal localization of the target mRNA and then its function can be determined by analyzing several axon-related properties such as the length of axon and 47 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC synaptic activity. The results will provide a list of candidate mRNAs whose axonal localization is crucial for axon growth and functions. Example 9 – Manipulation of RNA for axonal localization in vivo.

[0140] According to the present prophetic example, we will choose 100 mRNAs that show strong phenotypes for axon growth, guidance, or synaptic activity and validate directly in mice brain. Briefly, either via co-transfection of plasmids or co-transduction of AAVs or lentivirus encoding the CRISPR-TO components, these components can be delivered into the brains of mice. After + / - ABA treatment, the axonal morphology can be imaged and / or neuronal activity can be measured in mice. Example 10 – Machine learning for predicting RNA localization in different cell types and in disease.

[0141] With the advent of massively multiplexed single-molecule imaging technologies such as MERFISH, in situ RNA sequencing technologies such as ExSeq, and proximity labeling-based spatial transcriptome mapping technologies such as APEX-seq and CAP-seq, thousands of RNAs have been observed to localize in various subcellular compartments. However, there is not a comprehensive list of localized RNAs across cell types. According to this prophetic example, we can combine existing functional data with existing imaging or sequencing data to build a full bioinformatic database of localized RNAs in different cells. This can provide an atlas of spatial RNA organization in human and mouse cells. Based on the atlas, a deep learning framework can be generated to computationally analyze the patterns of RNA localization, and correlate these patterns with RNA binding proteins, the presence of conserved RNA motifs, cell type specificity, and cell state. This can allow us to build an unprecedented model that can suggest factors as determinants of RNA localization and provide us with an atlas of spatial RNA distribution in cells and good candidates to support functional studies in the following steps. Example 11 – Apply CRISPR-TO to explore the pathological mechanism and treatment of SMA disease in vivo.

[0142] Dysregulation of mRNA localization is correlated with many neurological diseases. However, a direct functional relationship between mRNA mis-localization and the pathology of diseases is missing. In this prophetic example, embodiments of the mRNA localization monitoring and manipulation framework according to the present disclosure to explore this question. Spinal 48 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC muscular atrophy (SMA), the biggest genetic cause of infant death and the most common form of neuromuscular disease in children and young adults, is an excellent target. SMA is characterized by the degeneration of spinal motor neurons and caused by reduced levels of the survival motor neuron (SMN) protein due to deletion or mutations in the SMN1 gene. It was reported that knockdown of SMN reduced the axonal localization of ~1,000 RNAs associated with axon growth and synaptic activity in primary embryonic mouse mot neurons, indicating that disrupted mRNA localization may contribute to the pathophysiology of SMA, including the defects in axon elongation and presynaptic differentiation. However, the contributions of these mis-localized transcripts to the phenotypes of SMA remained unclear.

[0143] According to the present prophetic example, for candidate mRNAs that induce SMA, the contributions of mRNAs to the phenotypes of SMN-deficient motor neurons can be explored, including defects in axon elongation and presynaptic differentiation. Specifically, it can be tested whether such perturbation leads to the phenotypes of SMN-deficient motor neurons. This framework can also be applied to recover the axonal localization of these mRNAs in SMN- deficient motor neurons and analyze whether this strategy can correct (or otherwise relieve) the defective phenotype[s]. Using a machine learning framework as discussed in Example 7 above, signatures of RNA sequence motifs and RNA-binding proteins can be analyzed to nail down potential contributors that can dysregulate native mRNA localization. It is thought that these experiments can uncover the relationship between the disrupted axonal mRNA localization and the functional defects of SMN-deficient motor neurons and find out the key mRNAs leading to the defected phenotypes. Example 12 – Treatment using CRISPR-TO as therapy to mitigate the symptom in SMA.

[0144] According to the present prophetic example, embodiments of CRISPR-TO can be applied to recover correct axonal localization in a SMA mouse model named SMNǻ7. It can be examined whether it can help to relieve the pathological symptom of the mice. The CRISPR-TO components (including targeting key mRNAs) can be delivered into the motor neurons of diseases mice by AAV or lentivirus in order to promote the axonal localization of the key mRNA. The symptoms of SMA, in addition to the animal viability, can be examined. Example 13 – Test more RNA-targeting CRISPR-Cas systems for CRISPR-TO.

[0145] We tested more CRISPR-Cas systems for CRISPR-TO by recruiting endogenous GAPDH mRNA to the outer mitochondrial membrane (OMM). Besides PspCas13b, we selected four RNA-guided RNA- 49 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC targeting Cas effectors, including type VI-D RfxCas13d (Konermann et al., 2018), type VI-A LwaCas13a (Abudayyeh et al., 2017), a compact Cas13bt1 (Kannan et al., 2021), and type III-E DiCas7-11 (Ozcan et al., 2021). Among them, dPspCas13b exhibited the highest RNA relocalization efficiency, while dRfxCas13d and dLwaCas13a were ineffective. These results indicate that different dCas proteins possess distinct RNA binding activities and can impact CRISPR-TO efficiency. Example 14 – CRISPR-TO enables manipulating the localization of multiple RNAs simultaneously.

[0146] To test whether CRISPR-TO can simultaneously manipulate the localization of multiple RNAs in primary neurons, we located endogenous Actb mRNA together with Gap43 mRNA which encodes a protein associated with axonal outgrowth and regeneration (Denny, 2006), to neurite tips using CRISPR-TO. We detected simultaneous mRNA localization at neurite tips only when co-expressing gRNAs targeting the two mRNAs. Omitting either gRNA resulted in the absence of the corresponding mRNA at the neurite tips, which further confirms the specificity of CRISPR-TO. Example 15 –mRNAs localized to neurite tips by CRISPR-TO exhibit effective local translation.

[0147] We next investigated whether mRNA localized to neurite tips by CRISPR-TO can be locally translated. We used a reporter mRNA expressing the photoconvertible fluorescent protein Dendra2 with 24 GCN4 repeats in the 3’ UTR. Dendra2 fluorescence irreversibly switches from green to red after UV illumination, allowing differentiation between newly translated proteins (green) and those produced before UV conversion (red) (Pilaz et al., 2023). We hypothesized that if the reporter mRNA localized to neurite tips can be locally translated, green fluorescence recovery at the tips after UV conversion would be faster. To test this, we transfected primary mouse cortical neurons with plasmids encoding CRISPR-TO targeting GCN4, CAAX-HaloTag, and the reporter mRNA, followed by ABA or DMSO treatment for 6 hours. After UV conversion, we monitored Dendra2 fluorescence recovery at neurite tips for 50 minutes. In the ABA group, green fluorescence increased steadily, while it decreased in the DMSO group (FIG. 3A-3B). The mean fluorescence recovery over the period across 72 neurite tips in the ABA group was significantly higher than the DMSO group (FIG. 3C), indicating active local translation. 50 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC Example 16 – CRISPR-TO co-transports ribosomes as passenger molecules with mRNAs from the soma to neurite tips.

[0148] The detection of ribosome-bound mRNA has been used to resolve translation (Zeng et al., 2023). To further investigate translation of endogenous mRNA localized by CRISPR-TO, we performed dual-color RNA FISH for Actb mRNA and 28S ribosomal RNA (rRNA), Rn28s1 (Antony et al., 2023; Buxbaum et al., 2014). In neurons treated with gT+ABA, we detected strong Rn28s1 rRNA signals co-localized with Actb mRNA at neurite tips (FIG.4A), indicating there are abundant ribosomes for local translation at neurite tips. Interestingly, in the neurites where Actb mRNA particles are sparsely distributed for precise characterization, we observed 66% of Actb mRNA particles co-localized with Rn28s1 rRNA and varying rRNA intensities on each mRNA particle, likely suggesting polysomes co-transported with mRNA (FIG. 4B). This data provides direct imaging evidence for one possible source of neuritic ribosomes (Fernandopulle et al., 2021) which co-transport as passenger molecules with mRNAs from the soma to neurite tips over long distances in neurons. It also suggests mRNA during transport may undergo translation and those located to neurite tips by CRISPR-TO can be actively translated with abundant ribosomes co- transported there (FIG.4C). Example 17 – CRISPR-TO enables manipulating the localization of noncoding RNA.

[0149] We next tested if CRISPR-TO can manipulate the localization of noncoding RNA (ncRNA). To do that, we fused PYL1 with TRF1, a core component of the telomere nucleoprotein complex (Munoz et al., 2009). We targeted telomeric repeat-containing RNA (TERRA), a long noncoding RNA (lncRNA) that partially localizes to telomeres for regulating telomere maintenance (Canale et al., 2023), and observed that CRISPR-TO further enhanced its localization to telomeres in HeLa cells. Example 18 – CRISPR-TO manipulates endogenous RNA localization in vivo.

[0150] To determine whether CRISPR-TO can manipulate endogenous RNA localization in vivo, we searched for an animal organ system that shows obvious polar RNA distribution with simple 2D imaging techniques. Thus, we turn to the retina in the visual system. After performing in situ RNAscope staining in mouse retina tissues, we found that photoreceptors (rod / cone) have bipolar Actb mRNA distribution where Actb mRNA is largely localized in the distal ends of the cells. As a result, we designed a CRISPR-TO 51 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC system using PYL1 fused with KIFC1 and dCas13 to ABI and co-delivered as DNA into retina cells to re- localize Actb mRNA in the proximal ends of the microtube toward the cell body. We then used electroporation to deliver the CRISPR-TO plasmids into the retinas of wild type mice on the date of birth. Twenty-one days after they were born, the mice were treated with ABA using intraperitoneal (IP) injection for 8 hours. Retinas of the animals were collected at the end of ABA treatment, and RNAscope was performed in tissues of all electroporated retinas.

[0151] Only in the experimental group (gT+ABA), a portion of Actb mRNA was localized to the center of the photoreceptor cells and Müller glial cells where electroporation occurred. We compared the experimental group with the two control groups (gNT+ABA and gT+DMSO), we found that 7 of 16 animals in the experimental group have Actb mRNA localization events, and none in both control groups. In conclusion, CRISPR-TO is able to alter endogenous RNA localization in living animals. REFERENCES OF THE PRESENT DISCLOSURE ^ Abil, Z., Gumy, L.F., Zhao, H., and Hoogenraad, C.C. (2017). Inducible control of mRNA transport using reprogrammable RNA-binding proteins. ACS Synth Biol 6, 950-956. Alon, S., Goodwin, D.R., Sinha, A., Wassie, A.T., Chen, F., Daugharthy, E.R., Bando, Y., Kajita, A., Xue, A.G., Marrett, K., et al. (2021). Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems. Science 371. Belmont, B.J., and Niles, J.C. (2012). 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SEQUENCES OF THE PRESENT DISCLOSURE Sequences of embodiments of the present disclosure are recited below: >PYL1 polypeptide CDS; protein_id="NP_001331305.1"; GeneID:”834722" (SEQ ID NO:1) 54 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGRCSSLL AQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRE RLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEG NSEEDTRLFADTVIRLNLQKLASITEAMNRNNNNNNSSQVR >Protein phosphatase 2C family protein (ABI1); protein_id="NP_194338.1"; GeneID:”828714" (SEQ ID NO:2) MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCSVSG SHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRTESRSLFE FKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGS QVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETV GSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQ WNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEE ACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNI SVVVVDLKPRRKLKSKPLN >C-terminal 31 amino acids of mitochondrial antiviral-signaling protein (MAVS) for outer mitochondrial membrane (OMM) (SEQ ID NO:3) RPSPGALWLQVAVTGVLVVTLLVVLYRRRLH >DDX6 protein (NCBI Reference Sequence: NP_001244120.1) for p-bodies (SEQ ID NO:4) MSTARTENPVIMGLSSQNGQLRGPVKPTGGPGGGGTQTQQQMNQLKNTNTINNGTQQQ AQSMTTTIKPGDDWKKTLKLPPKDLRIKTSDVTSTKGNEFEDYCLKRELLMGIFEMGWE KPSPIQEESIPIALSGRDILARAKNGTGKSGAYLIPLLERLDLKKDNIQAMVIVPTRELALQ VSQICIQVSKHMGGAKVMATTGGTNLRDDIMRLDDTVHVVIATPGRILDLIKKGVAKV DHVQMIVLDEADKLLSQDFVQIMEDIILTLPKNRQILLYSATFPLSVQKFMNSHLQKPYEI NLMEELTLKGVTQYYAYVTERQKVHCLNTLFSRLQINQSIIFCNSSQRVELLAKKISQLG YSCFYIHAKMRQEHRNRVFHDFRNGLCRNLVCTDLFTRGIDIQAVNVVINFDFPKLAET YLHRIGRSGRFGHLGLAINLITYDDRFNLKSIEEQLGTEIKPIPSNIDKSLYVAEYHSEPVE DEKP >G3BP1 protein (NCBI Reference Sequence: NP_005745.1) for stress granules (SEQ ID NO:5) MVMEKPSPLLVGREFVRQYYTLLNQAPDMLHRFYGKNSSYVHGGLDSNGKPADAVYG QKEIHRKVMSQNFTNCHTKIRHVDAHATLNDGVVVQVMGLLSNNNQALRRFMQTFVL APEGSVANKFYVHNDIFRYQDEVFGGFVTEPQEESEEEVEEPEERQQTPEVVPDDSGTFY DQAVVSNDMEEHLEEPVAEPEPDPEPEPEQEPVSEIQEEKPEPVLEETAPEDAQKSSSPAP ADIAQTVQEDLRTFSWASVTSKNLPPSGAVPVTGIPPHVVKVPASQPRPESKPESQIPPQR PQRDQRVREQRINIPPQRGPRPIREAGEQGDIEPRRMVRHPDSHQLFIGNLPHEVDKSELK DFFQSYGNVVELRINSGGKLPNFGFVVFDDSEPVQKVLSNRPIMFRGEVRLNVEEKKTR AAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGFGVGRGLAPRQ >TERF1 protein (NCBI Reference Sequence: NP_059523.2) for telomere (SEQ ID NO:6) MAEDVSSAAPSPRGCADGRDADPTEEQMAETERNDEEQFECQELLECQVQVGAPEEEE EEEEDAGLVAEAEAVAAGWMLDFLCLSLCRAFRDGRSEDFRRTRNSAEAIIHGLSSLTA CQLRTIYICQFLTRIAAGKTLDAQFENDERITPLESALMIWGSIEKEHDKLHEEIQNLIKIQ AIAVCMENGNFKEAEEVFERIFGDPNSHMPFKSKLLMIISQKDTFHSFFQHFSYNHMME 55 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC KIKSYVNYVLSEKSSTFLMKAAAKVVESKRTRTITSQDKPSGNDVEMETEANLDTRKSV SDKQSAVTESSEGTVSLLRSHKNLFLSKLQHGTQQQDLNKKERRVGTPQSTKKKKESRR ATESRIPVSKSQPVTPEKHRARKRQAWLWEEDKNLRSGVRKYGEGNWSKILLHYKFNN RTSVMLKDRWRTMKKLKLISSDSED >HSF1 protein (NCBI Reference Sequence: NP_005517.1) for nuclear stress bodies (SEQ ID NO:7) MDLPVGPGAAGPSNVPAFLTKLWTLVSDPDTDALICWSPSGNSFHVFDQGQFAKEVLP KYFKHNNMASFVRQLNMYGFRKVVHIEQGGLVKPERDDTEFQHPCFLRGQEQLLENIK RKVTSVSTLKSEDIKIRQDSVTKLLTDVQLMKGKQECMDSKLLAMKHENEALWREVAS LRQKHAQQQKVVNKLIQFLISLVQSNRILGVKRKIPLMLNDSGSAHSMPKYSRQFSLEH VHGSGPYSAPSPAYSSSSLYAPDAVASSGPIISDITELAPASPMASPGGSIDERPLSSSPLVR VKEEPPSPPQSPRVEEASPGRPSSVDTLLSPTALIDSILRESEPAPASVTALTDARGHTDTE GRPPSPPPTSTPEKCLSVACLDKNELSDHLDAMDSNLDNLQTMLSSHGFSVDTSALLDLF SPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPG SVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS >Truncated KIF5A protein (aa1-559) (NCBI Reference Sequence: NP_001034089.1) for anterograde transport on microtubules (SEQ ID NO:7) MAETNNECSIKVLCRFRPLNQAEILRGDKFIPIFQGDDSVIIGGKPYVFDRVFPPNTTQEQ VYHACAMQIVKDVLAGYNGTIFAYGQTSSGKTHTMEGKLHDPQLMGIIPRIARDIFNHI YSMDENLEFHIKVSYFEIYLDKIRDLLDVTKTNLSVHEDKNRVPFVKGCTERFVSSPEEIL DVIDEGKSNRHVAVTNMNEHSSRSHSIFLINIKQENVETEQKLSGKLYLVDLAGSEKVSK TGAEGAVLDEAKNINKSLSALGNVISALAEGTKSYVPYRDSKMTRILQDSLGGNCRTTM FICCSPSSYNDAETKSTLMFGQRAKTIKNTASVNLELTAEQWKKKYEKEKEKTKAQKET IAKLEAELSRWRNGENVPETERLAGEDSALGAELCEETPVNDNSSIVVRIAPEERQKYEE EIRRLYKQLDDKDDEINQQSQLIEKLKQQMLDQEELLVSTRGDNEKVQRELSHLQSEND AAKDEVKEVLQALEELAVNYDQKSQEVEEKSQQNQLLVDELSQKVATMLSLESELQRL QEVSGHQRKRIAEVLNGLMRDLS >Truncated KIF5B protein (aa1-555) (NCBI Reference Sequence: NP_032474.2) for anterograde transport on microtubules (SEQ ID NO:8) MADPAECNIKVMCRFRPLNESEVNRGDKYVAKFQGEDTVMIASKPYAFDRVFQSSTSQ EQVYNDCAKKIVKDVLEGYNGTIFAYGQTSSGKTHTMEGKLHDPEGMGIIPRIVQDIFN YIYSMDENLEFHIKVSYFEIYLDKIRDLLDVSKTNLSVHEDKNRVPYVKGCTERFVCSPD EVMDTIDEGKSNRHVAVTNMNEHSSRSHSIFLINVKQENTQTEQKLSGKLYLVDLAGSE KVSKTGAEGAVLDEAKNINKSLSALGNVISALAEGSTYVPYRDSKMTRILQDSLGGNCR TTIVICCSPSSYNESETKSTLLFGQRAKTIKNTVCVNVELTAEQWKKKYEKEKEKNKTLR NTIQWLENELNRWRNGETVPIDEQFDKEKANLEAFTADKDIAITSDKPAAAVGMAGSFT DAERRKCEEELAKLYKQLDDKDEEINQQSQLVEKLKTQMLDQEELLASTRRDQDNMQ AELNRLQAENDASKEEVKEVLQALEELAVNYDQKSQEVEDKTKEYELLSDELNQKSAT LASIDAELQKLKEMTNHQKKRAAEMM Truncated KIFC1 protein (aa125-673) (NCBI Reference Sequence: NP_002254.2) for retrograde transport on microtubules (SEQ ID NO:9) 56 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC MAGGKKPSKRPAWDLKGQLCDLNAELKRCRERTQTLDQENQQLQDQLRDAQQQVKA LGTERTTLEGHLAKVQAQAEQGQQELKNLRACVLELEERLSTQEGLVQELQKKQVELQ EERRGLMSQLEEKERRLQTSEAALSSSQAEVASLRQETVAQAALLTEREERLHGLEMER RRLHNQLQELKGNIRVFCRVRPVLPGEPTPPPGLLLFPSGPGGPSDPPTRLSLSRSDERRG TLSGAPAPPTRHDFSFDRVFPPGSGQDEVFEEIAMLVQSALDGYPVCIFAYGQTGSGKTF TMEGGPGGDPQLEGLIPRALRHLFSVAQELSGQGWTYSFVASYVEIYNETVRDLLATGT RKGQGGECEIRRAGPGSEELTVTNARYVPVSCEKEVDALLHLARQNRAVARTAQNERS SRSHSVFQLQISGEHSSRGLQCGAPLSLVDLAGSERLDPGLALGPGERERLRETQAINSSL STLGLVIMALSNKESHVPYRNSKLTYLLQNSLGGSAKMLMFVNISPLEENVSESLNSLRF ASKVNQCVIGTAQANRK >N-terminal 27 amino acids of endoplasmic reticulum (ER)-resident protein P450 oxidase 2C1 for outer ER membrane (SEQ ID NO:10) MDPVVVLGLCLSCLLLLSLWKQSYGGG >CAAX peptide for inner cell membrane (SEQ ID NO:11) GKKKKKKSKTKCVIM >myr peptide for inner cell membrane (SEQ ID NO:12) MGCIKSKENK >three tandem nucleolar targeting sequences from NF-kB-inducing kinase (NIK3x) for nucleolus (SEQ ID NO:13) SNSRMRKKRKKKLRILRMRKKRKKKLRILRMRKKRKKKLRIL >^KASH4 domain for outer nuclear membrane (SEQ ID NO:14) DPASRQPLTFLLILFLLFLLLVGAMFLLPASGGPCCSHARIPRTPYLVLSYVNGLPPV >Linker1 (L1) (SEQ ID NO:15) AAAGS >Linker2 (L2) (SEQ ID NO:16) EGALEALPVAT >Linker3 (L3) (SEQ ID NO:17) SGGSSGGSSGSETPGTSESATPESSGGSSGGS ***

[0152] Although the foregoing has been described in some detail by way of illustration and example, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference with respect to the material for which they are cited. 57 KILPATRICK TOWNSEND 789227821

Claims

Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC WHAT IS CLAIMED:

1. A cell, comprising: a heterologous intracellular molecular transport dimerization pair, the pair comprising: (i) a localization polypeptide, and (ii) a ribonucleic acid (RNA) carrier polypeptide.

2. The cell of claim 1, wherein the localization polypeptide comprises an amino acid sequence encoding a subcellular compartment polypeptide or a motor polypeptide.

3. The cell of claim 2, wherein the subcellular compartment polypeptide or motor polypeptide comprises a telomere polypeptide, a mitochondrial polypeptide, a endoplasmic reticulum polypeptide, a lysosomal polypeptide, a p-body polypeptide, a stress granule polypeptide, a TIS granule polypeptide, a peroxisomal polypeptide, a ribosomal polypeptide, a golgi apparatus polypeptide, a cytoskeletal polypeptide, a cystosolic polypeptide, a vacuole polypeptide, a cellular membrane polypeptide, an exosomal polypeptide, a nuclear polypeptide, or a nucleolar polypeptide.

4. The cell of claim 2, wherein the motor polypeptide comprises a myosin polypeptide, a kinesin superfamily polypeptide, a dynein polypeptide, a polymerization motor polypeptide, a rotary motor polypeptide, or a nucleic acid motor polypeptide.

5. The cell of any one of claims 2 to 4, wherein the localization polypeptide encodes a mitochondrial antiviral signaling polypeptide (MAVS), a translocase of the outer membrane (TOM) polypeptide, a ras GTPase-activating polypeptide-binding polypeptide 1 (G3BP1) polypeptide, a DEAD-Box Helicase 6 (DDX6) polypeptide, a truncated kinesin family member C1 (KIFC1), a truncated kinesin family member 5a (KIF5a), or a truncated kinesin family member 5b (KIF5b).

6. The cell of any one of claims 1 to 5, wherein the RNA carrier polypeptide comprises an amino acid sequence encoding a dead CRISPR-associated (dCas) polypeptide. 58 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 7. The cell of claim 6, wherein the dCas polypeptide is a type II, type IV, type III, or type VI CRISPR system dCas polypeptide.

8. The cell of claims 6 or 7, wherein the dCas polypeptide is a dCas13 polypeptide or dCas7- 11 polypeptide.

9. The cell of any one of claims 6 to 8, wherein the dCas13 is a dCas13a polypeptide, dCas13b polypeptide, dCas13c polypeptide, or dCas13d polypeptide.

10. The cell of any of claims 6 to 9, further comprising a guide RNA (gRNA) configured to specifically bind to (a) the RNA carrier polypeptide and (b) an intracellular RNA.

11. The cell of claim 10, wherein the intracellular RNA comprises a polynucleotide encoding GAPDH, ACTB, SDHC, SDHD, NDUFS2, Psd95, CamKIIa, Grin2b, CD47, BCL-2, CD274 / PDL1, p53, EGFR, GPCR, or TFRC / CD71.

12. The cell of any one of claims 1 to 11, wherein the localization polypeptide and RNA carrier polypeptide are translationally fused.

13. The cell of any of claims 1 to 12, wherein the localization polypeptide further comprises a first dimerization handle translationally fused with the subcellular compartment polypeptide or a motor polypeptide, and the RNA carrier polypeptide comprises a second dimerization handle translationally fused with the RNA carrier polypeptide, wherein the first and second dimerization handles bind to one another.

14. The cell of claim 13, wherein binding of the first and second dimerization handle is chemically or optically inducible.

15. The cell of claims 13 or 14, wherein 59 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC a. the first dimerization handle comprises a pyrabactin resistance 1-like (PYL) polypeptide, a first FKBP12 polypeptide, or a FKBP polypeptide; and b. the second dimerization handle comprises an ABI polypeptide, a second FKBP12 polypeptide, a FRB polypeptide, a calcineurin polypeptide, or a cyclosporin A polypeptide.

16. The cell of claims 13 or 14, wherein a. the first dimerization handle comprises an ABI polypeptide, a second FKBP12 polypeptide, a FRB polypeptide, a calcineurin polypeptide, or a cyclosporin A polypeptide; and b. the second dimerization handle comprises a PYL polypeptide, a first FKBP12 polypeptide, or a FKBP polypeptide, respectively.

17. The cell of claim 15 or 16, wherein the PYL polypeptide is an Arabidopsis thaliana PYL1 polypeptide.

18. The cell of any one of claims 15 to 17, wherein the ABI polypeptide is an Arabidopsis thaliana ABI polypeptide.

19. The cell of any one of claims 1 to 18, further comprising a first detectable label linked to the localization polypeptide or the RNA carrier polypeptide.

20. The cell of any one of claims 1 to 18, further comprising a second detectable label linked to the localization polypeptide or the RNA carrier polypeptide not linked to the first detectable label.

21. The cell of any one of claims 15 to 20, further comprising abscisic acid (ABA), causing the binding of (a) and (b) within the cell.

22. The cell of any one of claims 1 to 21, wherein the localization polypeptide is linked to the RNA carrier polypeptide by a linker. 60 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 23. The cell of any one of claims 1 to 22, wherein the cell is an animal cell.

24. The cell of any one of claims 1 to 23, wherein the cell is not a plant cell.

25. The cell of any one of claims 1 to 24, wherein the cell is a neuron, a glial cell, a fibroblast, a intestinal epithelial cell, a mesenchymal cell, a T cell, a cancer cell, a stem cell, or a cell from an immortalized cell line.

26. A method of localizing RNA in a cell, comprising: a. delivering nucleic acids encoding the localization polypeptide and RNA carrier polypeptide of any one of claims 1 to 24 into a cell; and. b. inducing dimerization of the localization polypeptide and RNA carrier polypeptide inside the cell, forming an intracellular molecular transport dimerization pair.

27. The method of claim 26, further comprising, after step (a) or (b): b. delivering a guide RNA to the cell.

28. The method of claim 26 or 27, wherein the inducing is optical or chemical induction.

29. The method of any one of claims 26 to 28, wherein the inducing is by delivering abscisic acid to the cell.

30. The method of any one of claims 26 to 29, wherein the cell is an animal cell.

31. The method of any one of claims 26 to 30, wherein the cell is not a plant cell.

32. The method of any one of claims 26 to 31, further comprising detecting the dimerization pair.

33. A nucleic acid encoding a localization polypeptide of any one of claims 1 to 25. 61 KILPATRICK TOWNSEND 789227821Patent Attorney Docket No.: 110221-1461136-010000WO Client Reference No.: CZB-282S-PC 34. A nucleic acid encoding a ribonucleic acid (RNA) carrier polypeptide of any one of claims 1 to 25. 62 KILPATRICK TOWNSEND 789227821

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