Enhanced targeted pseudouridylation with small nuclear rnas

Abstract

This disclosure provides methods and compositions to increase the efficiency of targeted coding RNA pseudouridylation by H / ACA snoRNA, by localizing the H / ACA snoRNA more to the nucleoplasm where pre-mRNAs are transcribed and processed into mature mRNAs versus the nucleolus, where H / ACA snoRNAs natively modify ribosomal RNAs.

Description

[0001] Atty. Dkt. No.: 114198-6910 ENHANCED TARGETED PSEUDOURIDYLATION WITH SMALL NUCLEAR RNAS CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 657,534, filed on June 7, 2024, the contents of which are incorporated herein by reference in their entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under HG004659 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Roughly 10-15% of genetic diseases are caused by premature termination codons (PTCs), namely mutated UGA, UAG, and UAA sequences that occur in the coding regions of mRNA transcripts that result in an early stop codon during translation and truncated protein product. Examples of PTC diseases include Cystic Fibrosis and Hurler’s syndrome. Protein- free RNA-targeting technologies such as engineered tRNAs, A-to-I RNA templates, and engineered H / ACA snoRNAs have all been used with varying levels of success to suppress PTCs. Engineered tRNAs have many off-targets, including at canonical stop codons, due to their limited codon targeting capacity. A-to-I RNA templates that recruit endogenous ADAR to convert adenosine to inosine have a strong preference for the UAG context, with limited activity for the other sequence contexts (UGA and UAA). Thus, a need exists in the art to mitigate these limitations. This disclosure satisfies this need and provides related advantages as well. Atty. Dkt. No.: 114198-6910 SUMMARY OF THE DISCLOSURE More recently, engineered H / ACA snoRNAs that convert uridine to pseudouridine have shown promise for their combined specificity and codon generalizability (1, 2). For example, prior technology is disclosed (WO 2019 / 191232 A2: “NUCLEIC ACID MOLECULES FOR PSEUDOURIDYLATION”) that claims the use of engineered H / ACA snoRNAs for this purpose. However, snoRNAs localize predominantly to the nucleolus, not to the nucleus where pre-mRNAs are transcribed and processed into mRNAs. To address the limitations of the prior art, Applicant created a nucleoplasm-localizing U7smOPT snRNA backbone fused to an H / ACA snoRNA. This fusion increases the efficiency of targeted coding RNA pseudouridylation by H / ACA snoRNA, presumably by localizing the H / ACA snoRNA more to the nucleoplasm where pre-mRNAs are transcribed and processed into mature mRNAs versus the nucleolus, where H / ACA snoRNAs natively modify ribosomal RNAs. Endogenous U small nuclear RNAs (U snRNAs) form RNA-protein complexes responsible for eukaryotic processing of pre-mRNA into mature mRNA. Previous studies have demonstrated the utility of guide-programmable U snRNAs in targeted exon inclusion and exclusion. Applicant investigated whether snRNAs can also enhance conversion of RNA bases over state-of-the-art RNA targeting technologies in human cells. When compared to adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs, it was found that guided A>I snRNAs consistently increase adenosine-to-inosine editing efficiency for genes with higher exon counts, perturb substantially fewer genes in the transcriptome, and localize more persistently to the nucleus where ADAR is expressed. A>I snRNAs can also edit pre- mRNA 3′ splice sites to promote splicing changes. Finally, snRNA fusions to H / ACA box snoRNAs (U>Ψ snRNAs) increase targeted RNA pseudouridylation efficiency. Altogether, Applicant’s results advance the protein-free RNA base conversion toolbox and enhance minimally invasive RNA targeting technologies to treat genetic diseases. Applicant’s disclosure enhances the coding RNA (pre-mRNA and mRNA) pseudouridylation efficiency of engineered H / ACA snoRNAs by fusing to them RNAs Atty. Dkt. No.: 114198-6910 containing nucleoplasm-localizing features, namely components of U snRNAs natively involved in eukaryotic RNA splicing. Several embodiments of this disclosure are shown in FIG. 4. FIG. 4A contains the design of a coding RNA-targeting H / ACA snoRNA fused to U7smOPT snRNA backbone with relevant sequence features highlighted. FIG. 4B shows the design of a dual-luciferase reporter for measuring suppression of the PTC present in CFTR. A U7-promoter-driven CFTR-targeting H / ACA snoRNA fused by g(8) linker to U7smOPT snRNA backbone (U> Ψ snRNA) increases PTC suppression by ~70% over CFTR-targeting H / ACA snoRNA alone. FIG. 4C contains an illustration of targeted amplicon CMC sequencing to detect relative pseudouridylation of coding RNA transcripts, as well as endogenous experimental data from H / ACA snoRNA vs. U> Ψ snRNA on three separate coding RNA targets (ACTB, EEF2, and RPS6). U> Ψ snRNA increases pseudouridylation efficiency on all three endogenous transcripts, with improvements ranging from ~10% to ~100%. Applicant’s disclosure provides several benefits. In one aspect the fusions are used to characterize the role of pseudouridylation on coding transcripts in eukaryotic cells. The fusions can also be used to model suppression of premature termination codon diseases in cellular and animal models. The fusions also can be used to suppress premature termination codons, which cause ~10-15% of genetic diseases. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A – 1C: Programmable U snRNAs edit A>I on endogenous human transcripts with stronger activity on target genes containing high exon counts. (FIG. 1A), Schematic of three different RNA-guided A>I base editors targeting an mRNA transcript with the same C-mismatch guide: U7smOPT, U1 snRNA, and cadRNA. (FIG. 1B), Editing percent performance by transfection in HEK293T cells of three A>I base editors on targets sites from various human genes. A>I overperformance significance vs. cadRNA: ***, ****, *****: p < 1e-3, 1e-4, 1e-5 (one-way ANOVA, Bonferroni correction for multiple comparisons). Error bars reflect standard error of mean. n = 3 biological replicates per condition. (FIG. 1C), Editing percent performance by transfection in HEK293T cells of U7smOPT snRNA and cadRNA A>I base editors on targets sites from human genes with Atty. Dkt. No.: 114198-6910 high exon counts. A>I overperformance significance vs. cadRNA: **, ****, *****: p < 1e-2, 1e-4, 1e-5 (one-way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. FIGS. 2A – 2C: A>I snRNAs perturb fewer genes than circularized ADAR- recruiting RNAs. (FIG. 2A), Scatterplots of differential gene expression analysis against empty control (pUC19) of cadRNA backbone vs. U7smOPT snRNA backbone for RAB7A- and DMD-targeting guides. Cutoffs for significance are |log2 (fold change)| > 0.5 and adjusted p-value < 0.05. Upregulated genes are colored in red, and downregulated genes are colored in blue. n = 2 biological replicates per condition. (FIG. 2B), Number of local splicing variation events from differential splicing analysis against empty control (pUC19) of cadRNA backbone vs. U7smOPT snRNA backbone for RAB7A- and DMD-targeting guides, with p-value < 0.05 and different thresholds of differential Percent Spliced In (dPSI) for events (left). 4-way Venn diagrams of the number of significantly upregulated and downregulated splicing factors represented in the significantly perturbed genes from a (right). n = 2 biological replicates per condition. (FIG. 2C), Counts of significant transcriptome A>I edits, both exonic and non-exonic, absent in both empty control (pUC19) condition replicates of cadRNA backbone vs. U7smOPT snRNA backbone for RAB7A- and DMD-targeting guides, with different edit fraction thresholds (1 = 100% editing). Edit count significance of U7smOPT vs. cadRNA: p < 5e-2 (one-way ANOVA). n = 2 biological replicates per condition. FIGS. 3A – 3F: A>I snRNAs localize to the nucleus, enabling more efficient editing of lncRNAs and pre-mRNAs. (FIG. 3A), Schematic of subcellular localization qPCR to determine nuclear:cytosolic enrichment of A>I snRNA and cadRNA A>I base editors (left). Results from subcellular localization qPCR of the A>I base editors with guides targeting three different genes, and the lncRNA NEAT1 as a positive control for the assay (right). Nuclear:cytosolic enrichment significance vs. cadRNA: *, ***: p < 5e-2, 1e-3 (one- way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. (FIG. 3B), Rolling circle amplification FISH (RCA FISH) schematic, representative images, and plots of both rolonies per cell and mean rolony distance to nucleus for GAPDH-targeting A>I snRNA and cadRNA. RCA FISH was performed in human U-2 Atty. Dkt. No.: 114198-6910 OS cells with 10x magnification images. Scale bar: 20 microns. Significance of differences: p < 1e-2 (one-way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. (FIG. 3C), Editing percent performance by transfection in HEK293T cells of A>I snRNA and cadRNA A>I base editors on lncRNAs. A>I overperformance significance vs. cadRNA: ***, ****: p < 1e-3, 1e-4 (one-way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. (FIG. 3D), Schematic of A>I snRNA targeting 3′ splice site of pre-mRNA. (FIG. 3E and FIG. 3F), A>I editing, RT-PCR gels, and quantified Percent Spiced In performance by transfection in HEK293T cells of A>I snRNAs, antisense snRNAs, cadRNAs, and empty control (pUC19) for (FIG. 3E) endogenous ADAR-sensitive and (FIG. 3F) CRISPR base editor-sensitive 3′ splice sites of three human genes. Significance of differences: *, **, ****, *****: p < 5e-2, 1e-2, 1e-4, 1e-5 (one-way ANOVA, Bonferroni correction for multiple comparisons). Error bars reflect standard error of mean. n = 3 biological replicates per condition. † Gel quantification not reliable due to low abundance of dominant band. FIGS. 4A – 4D: U>Ψ snRNAs demonstrate increased potency over engineered snoRNAs in pseudouridylation and premature termination codon suppression. (FIG. 4A), Design of engineered U>Ψ snRNA, with guided H / ACA snoRNA targeting an mRNA transcript fused by RNA linker to U7smOPT backbone (SEQ ID NOs: 7 (left) and 8 (right)). (FIG. 4B), Schematic of dual-luciferase reporter to quantify premature termination codon (PTC) suppression of CFTR mutant W1282X by tested U>Ψ snRNA designs (top). Luciferase ratio performance by co-transfection in HEK293T cells of various U>Ψ snRNA designs on PTC suppression dual-luciferase reporter (bottom). FLuc / RLuc significance vs. U6 promoter-driven CFTR guide H / ACA snoRNA without linker / tail and U7smOPT backbone: *****: p < 1e-5 (one-way ANOVA, Bonferroni correction for multiple comparisons). Error bars reflect standard error of mean. n = 4 biological replicates per condition. (FIG. 4C), Effective pseudouridylation performance by transfection in HEK293T cells of guided U>Ψ snRNAs vs. H / ACA snoRNAs on stop codon context sequences from three human genes. Pseudouridylation overperformance significance vs. snoRNA: *, ***: p < 5e-2, 1e-3 (one-way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. (FIG. 4D), Schematic of CFTR-W1282X-induced nonsense mediated mRNA decay (NMD) and 16HBE14o- lentiviral transduction experiment (left). Atty. Dkt. No.: 114198-6910 CFTR and PuroR (transgene) expression evaluated by qPCR, with both GAPDH and ANXA5 as housekeeping genes (right). Significance of expression difference: *, **, ***: p < 5e-2, 1e- 2, 1e-3 (one-way ANOVA, Bonferroni correction for multiple comparisons). Error bars reflect standard error of mean. n = 3 biological replicates for empty vector condition; n = 4 biological replicates for snoRNA and U>Ψ snRNA conditions. FIG. 5: Competing model test for U7smOPT snRNA-to-cadRNA performance ratio. Scatter plots and corresponding Pearson correlation coefficients (r) for three competing models of U7smOPT snRNA-to-cadRNA performance ratio: gene exon counts, gene lengths, and empirical gene mRNA nuclear export rates. n = 15 genes (all targets from FIG. 1B, 1C). FIG. 6: Principal Component Analysis (PCA) plot of RNA-guided A>I base editor RNA sequencing data. PCA plot of RNA sequencing samples analyzed in FIG. 2, with each of two replicates colored by sample (pUC19, RAB7A-targeting cadRNA, RAB7A- targeting U7smOPT snRNA, DMD-targeting cadRNA, and DMD-targeting U7smOPT snRNA). FIG. 7: Alignment of RNA-guided A>I base editor RNA sequencing reads to DMD gene. Alignment showing RNA sequencing read pile-up across DMD-targeting cadRNA and U7smOPT snRNA replicates at the guide target on DMD. FIG. 8: Pathway analysis of significantly perturbed genes by RNA-guided A>I base editors. 4-way Venn diagrams of the number of significantly downregulated and upregulated genes across conditions from FIG. 2 (top). Enriched pathway heatmap of downregulated and upregulated genes conserved across both cadRNA guides (bottom). FIG. 9: Differential splicing analysis of RNA-guided A>I base editor RNA sequencing data. Scatterplots of local splicing variations (LSVs) against empty control (pUC19) of cadRNA backbone vs. U7smOPT snRNA backbone for RAB7A- and DMD- targeting guides. Cutoffs for significance are p-value < 0.05 and various dPSI (differential Percent Spliced In) values used in FIG. 2. Atty. Dkt. No.: 114198-6910 FIG. 10: Editing performance of A>I snRNAs with U7 vs. U1 snRNA promoter. Editing percent performance by transfection in HEK293T cells of A>I snRNAs targeting three different genes and driven by either U7 or U1 snRNA promoter. Overperformance significance vs. U7 promoter: **, ***: p < 1e-2, 1e-3 (one-way ANOVA). Error bars reflect standard error of mean. n = 3 biological replicates per condition. FIG. 11: Splicing gels for A>I editing -targeted pre-mRNA of DENND4A, FBXL4, and PDE4DIP. Full RT-PCR gels for all replicates of experiments in FIG. 3E. n = 3 biological replicates per condition. FIG. 12: Splicing gels for A>I editing-targeted pre-mRNA of AHCY, CTNNA1, and HSF1. Full RT-PCR gels for all replicates of experiments in FIG. 3F. n = 3 biological replicates per condition. FIGS. 13A – 13E: Targeted amplicon CMC sequencing for evaluating endogenous targeted pseudouridylation. (FIG. 13A), Schematic of BID-Seq to infer pseudouridylation of endogenous targeted mRNA by deletion rate. (FIG. 13B), BID-Seq deletion rates on synthetic RNA standards (100% U and 100% Ψ at targeted base) for CFTR reporter locus. n = 1 technical replicate per condition. (FIG. 13C), Effective pseudouridylation performance by transfection in HEK293T cells of guided U>Ψ snRNAs with (g)8 and (c)8 linkers vs. H / ACA snoRNA on CFTR reporter. Pseudouridylation difference significance vs. snoRNA: ****, *****: p < 1e-4, 1e-5 (one-way ANOVA, Bonferroni correction for multiple comparisons). Error bars reflect standard error of mean. n = 4 biological replicates per condition. (FIG. 13D), Schematic of targeted amplicon CMC sequencing to infer pseudouridylation of endogenous targeted mRNA by mutation / deletion rate. (FIG. 13E), Targeted amplicon CMC sequencing mutation / deletion rates on synthetic RNA standards (100% U and 100% Ψ at targeted base) for endogenous ACTB, EEF2, and RPS6 loci. n = 2 technical replicates per condition. FIGS. 14A – 14B: Experiments to evaluate mechanism of enhanced pseudouridylation by U>Ψ snRNAs. (FIG. 14A), Rolling circle amplification FISH (RCA FISH) schematic, representative images, and plots of both rolonies per cell and mean rolony distance to nucleus for EEF2-targeting H / ACA box snoRNA and U>Ψ snRNAs. RCA FISH Atty. Dkt. No.: 114198-6910 was performed in human U-2 OS cells with 10x magnification images. Scale bar: 20 microns. (FIG. 14B), Guide-specific qPCR to quantitate expression levels of H / ACA box snoRNA and U>Ψ snRNA constructs targeting CFTR, ACTB, EEF2, and RPS6. Significance of difference: ***: p < 1e-3 (one-way ANOVA). Error bars reflect standard error of mean. DETAILED DESCRIPTION Definitions The following definitions are intended to support and describe the embodiments and aspects of this disclosure, including as described in the attached Appendices, incorporated herein by reference. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. It must be noted that as used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of cells. As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series; Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Atty. Dkt. No.: 114198-6910 Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Techique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (Cold Spring Harbor Laboratory Press (2002)); Sohail ed. (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press). “Targeted pseudouridylation” is a process where uridine residues in specific RNA sequences are converted to pseudouridylation through RNA-directed modification. It is a gene-specific approach that suppresses nonsense-mediated mRNA decay (NMD) NMD and concurrently promotes premature termination codons (PTC) readthrough. An “engineered molecule or polynucleotide” intends a molecule or polynucleotide that has been modified or designed by humans through various techniques to have specific properties or functions. As used herein, the term “connecting” intends to bring to together or join distinct or separated molecules or sequences. “Ribosomal RNA” (rRNA) intends a type of non-coding RNA which is the primary component of ribosomes, which carries out protein synthesis in ribosomes. “Pre-mRNA” is or precursor mRNA, is the initial RNA transcript made from a gene during transcription. It's a molecule that contains both the coding regions (exons) and non- coding regions (introns) of the gene. Before it can be used for protein synthesis, pre-mRNA must undergo processing to remove the introns and create the mature mRNA. This processing includes splicing, capping and polyadenylation. Atty. Dkt. No.: 114198-6910 “mRNA” or “messenger RNA” is It's a single-stranded RNA molecule that carries the genetic instructions from DNA to the ribosome, where proteins are synthesized. Ribosomes read the mRNA sequence and translate it into a specific sequence of amino acids, which then folds into a protein. mRNA is essentially a copy of the DNA gene sequence, allowing for the genetic information to be accessed and used in the cytoplasm. “Long noncoding RNA” (lncRNA) intends long RNA transcripts, typically longer than 200 nucleotides, that do not code for proteins. They are transcribed from various genomic regions by RNA polymerases and are involved in a wide range of cellular processes, including gene regulation, chromatin organization, and cell development. LncRNAs can interact with DNA, RNA, and proteins, and they can regulate gene expression at multiple levels. “Enhancer RNA” is an example of a lncRNA transcribed from an enhancer region of the genome. These can play a role in regulating gene expression. “RNA found in the nucleoplasm of cells that is not ribosomal RNA” include for example, messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). Sn RNA intends a member of a small class of RNA molecules that are found within the splicing speckles and Cajal bodies of the cell nucleus. Wild-type and engineered of snRNA for use in gene editing are known in the art, e.g., US Application Publ. No.: 2024 / 0011028 A9, incorporated herein by reference. “U snRNA” intends RNA molecules found in the nucleus of eukaryotic cells that play a crucial role in pre-mRNA splicing, the process by which introns are removed from pre- messenger RNA to form mature RNA. Non-limiting examples include without limitation U1, U2, U4, U5, U6 and U7. Wild-type and engineered of U snRNA for use in gene editing are known in the art, e.g., US Application Publ. No.: 2024 / 0011028 A9, incorporated herein by reference. H / ACA box snoRNPs also referred to herein as H / ACA sno RNAs (ribonucleoproteins) are small ribonucleoprotein complexes, composed of a guide RNA and four core proteins, that guide the modification of uridine to pseudouridine in RNA, specifically ribosomal RNA, during the ribosome biogenesis process. They consist of a guide Atty. Dkt. No.: 114198-6910 snoRNA (a small, non-coding RNA) and four core proteins: Nop10, Nhp2, Gar1, and the pseudouridine synthase Cbf5 (Dyskerin in humans). They are non-coding RNA molecules. As used herein, the term U>Ψ snRNAs intends an U snRNA that has been modified through linking or fusion with H / ACA box snoRNAs. Examples of such are provided herein. The term “gRNA” or “guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction. Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. (2014) Nature biotechnology; Graham, D., et al. (2015) Genome Biol. “Single guide RNA” or “sgRNA” is a specific type of gRNA that combines tracrRNA (transactivating RNA), which binds to Cas9 to activate the complex to create the necessary strand breaks, and crRNA (CRISPR RNA), comprising complimentary nucleotides to the tracrRNA, into a single RNA construct. “U snRNA” (U1, U2, U4, U5, U6) are small nuclear RNAs that are essential components of the spliceosome that remove introns from pre-mRNA. These RNAs are involved in recognizing and removing introns from pre-mRNA, a crucial step in gene expression. They are also involved in RNA processing and regulation. U7smOPT snRNA (SEQ ID NO: 5) is a modified U7 snRNA sequence as shown below. The underlined nucleotides represent modified nucleotides from wildtype U7 snRNA: AGAATTTTTGGAGTAGGCTTTCTGGCTTTTTACCGGAAAGCCCCT A “RNA linker” is a short sequence of nucleotides (either RNA or DNA) used to connect two RNA molecules or to connect RNA to another molecule, such as a protein or a bead. In one aspect, the connection is a covalent bond. In one aspect, the RNA linker or DNA, is from about 1 to about 20 nucleotides in length, or alternatively from about 3 to about 20 nucleotides in length, or about 1 to about 15 nucleotides in length, or about 3 to about 15 nucleotides in length, or about 3 to about 10 nucleotides in length, or about 1 to about 10 nucleotides in length, or about 3 to about 8 nucleotides in length, or about 1 to about 8 nucleotides in length, or about 3 to about 5 nucleotides in length, or about 8 nucleotides in length. In a further aspect, the linker comprises at least 70% of the nucleotides selected from guanines, cytosines, or a mixture of both across the length of the linker. In a further aspect, the linker is from about 1 to about 20 nucleotides in length, or alternatively from about 3 to Atty. Dkt. No.: 114198-6910 about 20 nucleotides in length, or about 1 to about 15 nucleotides in length, or about 3 to about 15 nucleotides in length, or about 3 to about 10 nucleotides in length, or about 1 to about 10 nucleotides in length, or about 3 to about 8 nucleotides in length, or about 1 to about 8 nucleotides in length, or about 3 to about 5 nucleotides in length, or about 8 nucleotides in length, and the linker comprises at least 70% of the nucleotides selected from guanines, cytosines, or a mixture of both across the length of the linker. “Contacting” intends to bring into close proximity or location two previously separated or isolated molecules or composition. In one aspect, the term “contacting” intends mixing or creating a solution or mixture. In the context of in vivo use, the term can intend administration to a subject such as an animal, a mammal or a human patient. The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced there from. As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample. The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, biological, or cellular material and intend those having minimal homology while still maintaining desired structure or functionality. As used herein, the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular specified effect. As used herein, the term “gRNA” refers to a guide RNA sequence, known in the art to be used for example with gene targeting such as with the CRISPR-Cas9 system to facilitate targeting of the gene. gRNAs typically comprise a promoter, gRNA scaffold, and a target Atty. Dkt. No.: 114198-6910 specific sequence. Where more than one gRNA is present in a construct, spacers may be used to ensure gene targeting. The target specific sequences may be experimentally determined or found on one of many public databases, such as Addgene (www.addgene.org). gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA). In some aspects, a gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233: 74-83). Non-limiting examples of guide RNAs for use in the embodiments of this disclosure are shown in Table 2, and equivalents of each sequence shown in Table 2, across the full length of the sequence. The term “promoter” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include U1, U7, hU6 and mU6 promoter, CMV promoter, a T7 promoter, and EF- 1α promoter. Further, virus-derived promoters, some of which are noted above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters. In some embodiments, the promoter is coupled to an enhancer to increase the transcription efficiency. Non-limiting examples of enhancers include an RSV enhancer or a CMV enhancer. Non- limiting exemplary promoter sequences are provided herein below and provided in Table 4 below, as well as equivalents thereof across the full length of the polynucleotide: CMV promoter (SEQ ID NO: 1): ATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC Atty. Dkt. No.: 114198-6910 ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAA CCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAC CGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTT, or a biological equivalent thereof. U6 promoter (SEQ ID NO: 2): GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTA GAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA CGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTT AAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTT ATATATCTTGTGGAAAGGACGAAACACC, or a biological equivalent thereof. U1 promoter sequence (SEQ ID NO: 3): taacacaggctaaggaccagcttctttgggagagaacagacgcaggggcgggagggaaaaagggagaggcagacgtc acttccccttggcggctctggcagcagattggtcggttgagtggcagaaaggcagacggggactgggcaaggcactgtcggtgaca tcacggacagggcgacttctatgtagatgaggcagcgcagaggctgctgcttcgccacttgctgcttcaccacgaaggagttcccgtg ccctgggagcgggttcaggaccgctgatcggaagtgagaatcccagctgtgtgtcagggctggaaagggctcgggagtgcgcggg gcaagtgaccgtgtgtgtaaagagtgaggcgtatgaggctgtgtcggggcagaggcccaagatctc, or a biological equivalent thereof. U7 promoter sequence (SEQ ID NO: 4): ggcttaacaacaacgaaggggctgtgactggctgctttctcaaccaatcagcaccgaactcatttgcatgggctgagaaca aatgttcgcgaactctagaaatgaatgacttaagtaagttccttagaatattatttttcctactgaaagttaccacatgcgtcgttgtttataca gtaataggaacaagaaaaaagtcacctaagctcaccctcatcaattgtggagttcctttatatcccatcttctctccaaacacatacgcag, or a biological equivalent thereof. EF1α promoter(SEQ ID NO: 6): CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCG AGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGC GGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTG GGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGG Atty. Dkt. No.: 114198-6910 GTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGT ACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGC CTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCG CTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTC GATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTT GGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGG CGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGC CGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGG CAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCG GCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCG GGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCAT GTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTT TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCC CACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCT CCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACA GTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAG, or a biological equivalent thereof. The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10 %, 5 % or 1 %. Atty. Dkt. No.: 114198-6910 “Comprising” or “comprises” is intended to mean that the compositions, for example media, and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. As used herein, comparative terms as used herein, such as high, low, increase, decrease, reduce, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference. “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Atty. Dkt. No.: 114198-6910 “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. 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. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides. The term “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3′ and 5′ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure Atty. Dkt. No.: 114198-6910 or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In one aspect, an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods. In another aspect, an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher affinity to a reference antibody or antigen binding fragment. In another aspect, the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen tinder a competitive ELISA assay. In another aspect, an equivalent intends at least about 70%, or about 80% homology or identity and alternatively, at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences across the full length of the polynucleotide. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non- limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non- redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address:align.genome.jp, last accessed on Mar. 7, 2011. Atty. Dkt. No.: 114198-6910 “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions. “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. Examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C.; hybridization buffer concentrations of about 6×SSC to about 10×SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C.; buffer concentrations of about 9×SSC to about 2×SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation Atty. Dkt. No.: 114198-6910 times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for. “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, e.g., HEK 293 cells or HEK 293T cells. “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called Atty. Dkt. No.: 114198-6910 on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 μm in diameter and 10 μm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium. The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose. The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non- human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated peptide fragment” is meant to include peptide fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. Atty. Dkt. No.: 114198-6910 As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the composition or cell to provide the therapeutic benefit in vitro or in vivo by at least 10%, 25%, 40%, 60%, 80%, 90% or 95% as compared to control. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Administration or delivery in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective Atty. Dkt. No.: 114198-6910 means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell, solid tumor or cancer being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. The pharmaceutical compositions can be administered by inhalation, orally, intranasally, parenterally, injection, orally and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present disclosure, the compositions can also contain other pharmaceutically active compounds or a plurality of systems or cells of the disclosure. More particularly, an agent of the present disclosure also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. While it is possible for the agent to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising, or consisting essentially of, or consisting of at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing Atty. Dkt. No.: 114198-6910 into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate. Formulations suitable for nasal administration or aerosol (directly into the lung), wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered as a dry powder or in an inhaler device by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the agent. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and Atty. Dkt. No.: 114198-6910 may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this disclosure may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this disclosure be combined with other suitable compositions and therapies. The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, hybrid polynucleotides or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., WO 97 / 03211 and WO 96 / 39154. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs or those as described herein. 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. As used herein, a "vector" refers to a construct which is capable of delivering, and, in some embodiments expressing, a polynucleotide in to a cell. Non-limiting examples of delivery vectors include viral vectors, nucleic acid expression vectors (such as a plasmid), Atty. Dkt. No.: 114198-6910 naked DNA, and certain eukaryotic cells (e.g., producer cells). In some embodiments, nucleic acids described by the disclosure are delivered via a viral vector. Examples of viral vectors include retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD 100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV, HSV-1, HSV-2), as described by Chira et al. (2015) Oncotarget, 6(31): 30673- 30703. In some embodiments, nucleic acids described by the disclosure are delivered by an adeno-associated virus (AAV) vector (e.g., a recombinant AAV (rAAV) vector). The terms “non-naturally occurring” or “engineered” or “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions Atty. Dkt. No.: 114198-6910 are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y. Where reference is made to a polynucleotide sequence, then complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridising to the reference sequence under highly stringent conditions. Generally, in order to maximize the hybridization rate, relatively low-stringency hybridization conditions are selected: about 20 to 25° C lower than the thermal melting point (T m ). The T m is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH. Generally, in order to require at least about 85% nucleotide complementarity of hybridized sequences, highly stringent washing conditions are selected to be about 5 to 15° C lower than the T m . In order to require at least about 70% nucleotide complementarity of hybridized sequences, moderately-stringent washing conditions are selected to be about 15 to 30° C lower than the T m . Highly permissive (very low stringency) washing conditions may be as low as 50° C below the Tm, allowing a high level of mis-matching between hybridized sequences. Those skilled in the art will recognize that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences. Exemplary highly stringent conditions comprise incubation in 50% formamide, 5×SSC, and 1% SDS at 42° C, or incubation in 5×SSC and 1% SDS at 65° C, with wash in 0.2×SSC and 0.1% SDS at 65° C. “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence. Atty. Dkt. No.: 114198-6910 As used herein, the term “genomic locus” or “locus” (plural loci) is the specific location of a gene or DNA sequence on a chromosome. A “gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms. For the purpose of this disclosure, it may be considered that genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. As used herein, “expression of a genomic locus” or “gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product. The products of gene expression are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is functional RNA. The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses to generate functional products to survive. As used herein “expression” of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, “expression” also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. As described in aspects of this disclosure, sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Atty. Dkt. No.: 114198-6910 The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel (1990), GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue- specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal- dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al (1985), Cell, 41:521-530], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R- U5’ segment in LTR of HTLV-I (Mol. Cell. Biol. (1988), Vol. 8(1), p. 466-472,); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (1981) (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., gene-editing system transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). Atty. Dkt. No.: 114198-6910 Vectors can be designed for expression of gene-editing system transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, gene-editing system transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel (1990), GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990), GENE Atty. Dkt. No.: 114198-6910 EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. 60-89). In some embodiments, a vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). In some embodiments, a vector drives protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., (1983) Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, (1989) Virology 170: 31- 39). In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman, et al., (1987) EMBO J. 6: 187-195). When used in mammalian cells, the expression vector’s control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL. 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al.,(1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, (1988). Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, (1989) EMBO J. 8: 729-733) Atty. Dkt. No.: 114198-6910 and immunoglobulins (Baneiji, et al., (1983) Cell 33: 729-740; Queen and Baltimore, (1983). Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, (1989). Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., (1985), Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, (1990). Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, (1989). Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, (1989), Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, (1990), Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication dates of the reviews because it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, (1988), pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, (1974) Comprehensive Virology 3: 1-61). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Atty. Dkt. No.: 114198-6910 An “AAV vector” as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. An “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.” Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al. (1983) J. Virol., 45: 555-564; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al. (2004), J. Virol., 78: 6381-6388; the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology (2004), 330(2): 375-383. The sequence of the AAV rh.74 genome is provided in U.S. Patent 9,434,928, incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, pl9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The Atty. Dkt. No.: 114198-6910 two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, (1992) Current Topics in Microbiology and Immunology, 158: 97- 129. AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV- infected cells are not resistant to superinfection. Multiple studies have demonstrated long-term (> 1.5 years) recombinant AAV- mediated protein expression in muscle. See, Clark et al., (1996) Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087; and Xiao et al., (1996) J Virol, 70: 8098-8108. See also, Chao et al., Mol Ther, (2000), 2:619-623 and Chao et al., (2001) Mol Ther, 4:217-222. Moreover, because muscle is highly vascularized, recombinant Atty. Dkt. No.: 114198-6910 AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., (1997) Proc Natl Acad Sci USA, 94: 5804-5809 and Murphy et al., (1997) Proc Natl Acad Sci USA, 94: 13921- 13926. Moreover, Lewis et al., (2002) J Virol, 76: 8769-8775demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. Recombinant AAV (rAAV) genomes of the disclosure comprise, or consist essentially of, or yet further consist of a nucleic acid molecule encoding a payload and one or more AAV ITRs flanking the nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV- 12, AAV-13 and AAV rh74. Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., (2014), Molecular Therapy, 22(11): 1900-1909. The nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle specific expression, AAV1, AAV5, AAV6, AAV8 or AAV9 may be used. “Liposomes” are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C═C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), Atty. Dkt. No.: 114198-6910 dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloteoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1- carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, “Liposomes and Their Use in Biology and Medicine,” December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs). The polynucleotides can be encapsulated in such for administration in accordance with the methods described herein. A “micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle). Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue. Also included as a micelles are lipid nanoparticles. In some embodiments, a regulatory element is operably linked to one or more elements of a gene-editing system so as to drive expression of the one or more elements of the gene-editing system. Atty. Dkt. No.: 114198-6910 The molecules or polynucleotides described herein can further comprise one or more labels or detection tags (e.g., FLAG™ tag, epitope or protein tags, such as myc tag, 6 His, and fluorescent fusion protein). A “composition” is intended to mean a combination of active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g. a detectable label) or active (e.g. a gene delivery vehicle) alone or in combination with a carrier which can in one embodiment be a simple carrier like saline or pharmaceutically acceptable or a solid support as defined below. A “composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. A pharmaceutical composition intends that the carrier is suitable for administration to an animal, such as a human patien. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975), Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton). As used herein, the term “detectably labeled” means that the agent (biologic or small molecule) is attached to another molecule, compound or polymer that facilitates detection of the presence of the agent in vitro or in vivo. A “detectable label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g.,115Sn,117Sn and119Sn, a non-radioactive isotopes such as13C and15N, polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or Atty. Dkt. No.: 114198-6910 composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, luminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. In one aspect, the label encodes a luciferase polypeptide, e.g., a Renulla luciferase or a Firefly luciferase. Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996), Handbook of Fluorescent Probes and Research Chemicals (6thed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. 1996), Handbook of Fluorescent Probes and Research Chemicals (6thed.). Modes For Carrying Out the Disclosure Recently the gene editing field has turned from CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and other exogenous protein-encoded multicomponent systems toward minimally invasive single-component guided RNA scaffolds that recruit highly expressed endogenous protein machinery to edit genes at the RNA level1. Atty. Dkt. No.: 114198-6910 Researchers have particularly focused on suppressing in-frame premature termination codons (PTCs) caused by single base-pair substitution nonsense mutations in coding regions of mRNA transcripts. PTCs, which account for an estimated 10-15% of human genetic diseases such as Cystic fibrosis and Hurler syndrome, lead to truncated proteins and subsequent degradation of PTC-harboring mRNAs by nonsense mediated mRNA decay2. Given a clearly defined mechanism, several minimally invasive strategies already exist to treat PTC-associated diseases. More clinically established drugs like splice-switching antisense oligonucleotides and small molecules could be administered to patients3-5, but not all PTC diseases are amenable to exon skipping and PTC suppressor small molecules lack target site specificity. Similarly, engineered suppressor tRNAs designed to read through PTCs at the translational level could do so at any targeted stop codon context sequence6, 7. In contrast to these approaches, programmable guided RNA scaffolds that recruit highly expressed endogenous proteins to edit PTC bases directly strike a safer balance between minimal invasiveness and specificity. One such class of systems recruits endogenous adenosine deaminase acting on RNA (ADAR) enzymes to edit PTC adenosines to inosines (A>I). In mammalian cells, active ADAR family members ADAR1 and ADAR2 recognize regions of nuclear double stranded RNA (dsRNA), primarily at Alu repetitive regions but also in coding regions and even at splice sites8. After A>I editing, splicing and translation machinery generally recognizes inosine as its structurally comparable base, guanosine (G). Leveraging this finding, several groups have encoded a cytosine (C)-mismatch guided RNA scaffold of both linear and circular form which, when hybridized to the RNA sequence surrounding a targeted adenosine, recruits endogenous ADARs that efficiently edit the targeted adenosine opposite the cytosine to inosine9-11. While robust editing is possible, ADARs display a strong preference for the UAG motif, with diminished activity for the other PTC sequence contexts of UGA and UAA, in addition to varying by cell type expression12. Another class of systems utilizes H / ACA box snoRNPs, ribonucleoproteins highly conserved across eukaryotes which catalyze uridine-to-pseudouridine (U>Ψ) editing on snRNAs, ribosomal RNAs (rRNAs), and some mRNAs13, 14. In mammalian cells, H / ACA snoRNAs recruit four core proteins, DKC1, NOP10, NHP2, and GAR1. Together, these Atty. Dkt. No.: 114198-6910 proteins edit U>Ψ at a site between two guide-templated regions specified by the H / ACA snoRNA. Building upon initial work performed in yeast15, two groups reprogrammed human H / ACA snoRNAs to edit U>Ψ at all three PTC sequence contexts (UAG, UGA, and UAA), leading to successful translational readthrough16, 17. While a promising approach, snoRNA localizes predominantly to the nucleolus, and not to the nucleoplasm where pre-mRNAs are transcribed and processed into mRNAs, thus limiting its base editing potential. As disclosed herein, Applicant selected as a putative RNA nucleoplasm localization signal components of endogenous Uridine-rich small nuclear RNAs (U snRNAs), which natively recruit splicing machinery to process pre-mRNA into mature mRNA20, 21. As disclosed herein, Applicant evaluated the capacity of U snRNAs to enhance endogenous protein-mediated base editing, both A>I and U>Ψ, on mammalian coding transcripts. Targeted Pseudouridylation Molecules or Polynucleotides, Host Cells and Compositions Applicant provides herein a targeted pseudouridylation molecule or polypeptide comprising or consisting essentially of or consisting of: an engineered H / ACA box snoRNA polynucleotide with or linked to one or more guide RNA, e.g. a molecule or RNA targeting a eukaryotic RNA transcript; one or more U snRNA components or polynucleotides; and one or more RNA linker sequences linking or connecting the engineered H / ACA box snoRNA to the U snRNA components. In one aspect, the targeted pseudouridylation molecule or polynucleotide further comprises a detectable or purification label or tag. In one aspect, the targeted pseudouridylation molecules or polynucleotides are comprised of components or molecules for expression in animal and mammalian systems, e.g., mammalian cells and mammals such as humans and human cells. Non-limiting examples of engineered H / ACA box snoRNA polynucleotides are known in the art (see PCT / US2019 / 024282, incorporated herein by reference). Additional sequences are shown in FIG. 4 and Table 1, as portions of larger polynucleotides. In one aspect, the targeted eukaryotic RNA transcript comprises or includes, but is not limited to, pre-mRNA, mRNA, long noncoding RNA, enhancer RNA, or any other RNA found in the nucleoplasm of cells that is not a ribosomal RNA. Atty. Dkt. No.: 114198-6910 In another aspect, the one or more U snRNA components or polynucleotides are 20- 100bp sequences with >70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% seqiemce identity or homology across the full length of the polynucleotide to sequences derived from U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, U7 snRNA, U7smOPT snRNA (e.g., SEQ ID NO: 5), U11 snRNA, and / or U12 snRNA, or equivalents of each thereof. In another aspect, the one or more U snRNA polynucleotides comprises a U7smOPT snRNA polynucleotide of SEQ ID NO: 5, or an equivalent thereof having at least 70 percent sequence identity across the full length of the polynucleotide to SEQ ID NO: 5. In a further aspect, the U snRNA components are connected or linked to the 5′ end of the engineered H / ACA snoRNA, 3′ end of the engineered H / ACA snoRNA, or a mixture of both. Examples of linkers are provided herein. In a yet further aspect, the one or more RNA linkers comprise a sequence of guanines, cytosines, or a mixture of both. In one aspect, the connection is a covalent bond. In one aspect, the RNA linker is from about 1 to about 20 nucleotides in length, or alternatively from about 3 to about 20 nucleotides in length, or about 1 to about 15 nucleotides in length, or about 3 to about 15 nucleotides in length, or about 3 to about 10 nucleotides in length, or about 1 to about 10 nucleotides in length, or about 3 to about 8 nucleotides in length, or about 1 to about 8 nucleotides in length, or about 3 to about 5 nucleotides in length, or about 8 nucleotides in length. In a further aspect, the linker comprises at least 70% of the nucleotides selected from guanines, cytosines, or a mixture of both across the length of the linker. In a further aspect, the linker is from about 1 to about 20 nucleotides in length, or alternatively from about 3 to about 20 nucleotides in length, or about 1 to about 15 nucleotides in length, or about 3 to about 15 nucleotides in length, or about 3 to about 10 nucleotides in length, or about 1 to about 10 nucleotides in length, or about 3 to about 8 nucleotides in length, or about 1 to about 8 nucleotides in length, or about 3 to about 5 nucleotides in length, or about 8 nucleotides in length, and the linker comprises at least 70% of the nucleotides selected from guanines, cytosines, or a mixture of both across the full length of the linker. In one aspect, the targeted pseudouridylation molecules or polynucleotides are further comprised of elements such as a promoter sequence, a termination sequences or other components for expression in mammalian systems, e.g., mammalian cells and mammals such Atty. Dkt. No.: 114198-6910 as humans and human cells. Examples of polynucleotides comprising: a promoter, a termination sequence, and an H / ACA sno RNA polynucleotide (also referred to herein as the “backbone”) are provided in Table 1, e.g., SEQ ID NOs: 9-14Also shown in Table 1, additional elements not shown are referenced in general by <XXX>, e.g., where <guide> is a location of the linkage of the guide RNA to the backbone (identified as <guide>) in the table. The one or more guide RNAs are selected to target a eukaryotic RNA transcript. In one aspect, the guide RNA targets a CFTR polypeptide or a myosin polypeptide. In a further aspect the guide RNA is selected from a sequence shown in Table 2, e.g., SEQ ID NOs: 16- 28. In a further aspect, the gRNA is linked to the H / ACA box snoRNA polynucleotide and the one or more U snRNA polynucleotides fusion at a location internal to the fusion, e.g., not linked to the 5’ or 3’ end of the fusion. Applicant further provides isolated polynucleotide encoding the targeted pseudouridylation molecule or polynucleotide as described above, optionally wherein the polynucleotide is deoxyribonucleic acid (DNA) or further optionally wherein polynucleotide is expressed from an snRNA promoter, optionally selected from a U1 promoter or aU7 promoter. In one aspect, the polynucleotide further comprises a detectable or purification label or tag. In one aspect, the targeted pseudouridylation molecules or polynucleotides are comprised of components to be expressed in mammalian systems, e.g., mammalian cells and mammals such as humans and human cells. This disclosure further provides a vector or an isolated host cell comprising the isolated polynucleotide and / or pseudouridylation molecule or polynucleotide as described above. In one aspect, vector is selected from a plasmid, a viral vector, an adenoviral associated vector (AAV), a micelle, a lipid or a lipid nanoparticle (LNP). In a further embodiment, the vector is a lipid nanoparticle. The isolated host cell can be a prokaryotic or eukaryotic cell. In one embodiment, the host cell is a eukaryotic cell. In one aspect, the eukaryotic cell is an animal cell or a human cell, e.g., a HEK293 T cell, that optionally contains a genetic defect that optionally results in a disease, for example muscular dystrophy or cystic fibrosis. Also provided are compositions comprising a carrier and one or more of the isolated polynucleotides and / or pseudouridylation molecule as described above. In one aspect, the Atty. Dkt. No.: 114198-6910 carrier is a pharmaceutically acceptable carrier. In one aspect, vector is selected from a plasmid, a viral vector, an adenoviral associated vector (AAV), a micelle, a lipid or a lipid nanoparticle (LNP). In a further embodiment, the vector is a lipid nanoparticle. The host cell can be a prokaryotic or eukaryotic cell. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a eukaryotic cell. In one aspect, the eukaryotic cell is an animal cell or a human cell that optionally contains a genetic defect that optionally results in a disease, for example muscular dystrophy or cystic fibrosis. Methods of Use In one aspect this disclosure provides a method for delivering a pseudouridylation molecule or polynucleotide to a cell, the method comprising, or consisting essentially of, or yet further consisting of contacting the cell with an engineered H / ACA snoRNA or polynucleotide as described above. In one aspect, the engineered H / ACA snoRNA polynucleotide with a guide targeting a eukaryotic RNA transcript; one or more U snRNA components or polynucleotide; and one or more RNA linker sequences or polynucleotide connecting the engineered H / ACA snoRNA to U snRNA components. In one aspect, the targeted eukaryotic RNA transcript includes, but is not limited to, pre-mRNA, mRNA, long noncoding RNA, enhancer RNA, or any other RNA found in the nucleoplasm of cells that is not a ribosomal RNA. In another aspect, the one or more U snRNA components are 20- 100bp sequences with >70% or 75%, or 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% identity or homology to sequences derived from U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, U7 snRNA, U7smOPT, U11 snRNA, and / or U12 snRNA. In a further aspect, the U snRNA components are connected to the 5′ end of the engineered H / ACA snoRNA, 3′ end of the engineered H / ACA snoRNA, or a mixture of both. In another embodiment, the one or more RNA linkers comprise a sequence of guanines, cytosines, or a mixture of both. In a further aspect, the molecules further comprise a detectable or purification label. In one aspect of this method, the increased targeted pseudouridylation molecule or polynucleotide is delivered to a eukaryotic cell, such as an animal or human cell. In a further aspect, the increased targeted pseudouridylation molecule or polynucleotide is delivered to an animal, such as a human. Atty. Dkt. No.: 114198-6910 Delivery can be by any appropriate means, for example with use of vector. Non- limiting examples of vectors include a plasmid, a viral vector, an adenoviral associated vector (AAV), a micelle, a lipid or a lipid nanoparticle (LNP). In a further aspect, the increased targeted pseudouridylation molecule is delivered by lipid nanoparticle. In a yet further aspect, the increased targeted pseudouridylation molecule or polynucleotide is genetically encoded and expressed from an snRNA promoter, including, but not limited to, U1 or U7. Further provided are methods to deliver a targeted pseudouridylation molecule or polynucleotide to a cell comprising contacting the cell with the isolated targeted pseudouridylation molecules and / or the isolated polynucleotides and / or the vectors and / or the compositions as described herein. The contacting can be in vitro or in vivo. In one aspect, the cell is a eukaryotic cell, optionally a eukaryotic cell is an animal or a human cell. In one aspect, the cell contains a genetic defect or mutation that can result in a disease, such as for example muscular dystrophy. In another aspect, the cells express premature termination codons, which cause ~10-15% of genetic diseases. Applicant also discloses herein a method to deliver a targeted pseudouridylation molecule or polynucleotide to an animal comprising administering to the animal the isolated targeted pseudouridylation molecules and / or the isolated polynucleotides, and / or the vectors, and / or the host cells, and / or the compositions as described herein. In one aspect, the animal contains a genetic defect or mutation that can result in a disease, such as for example muscular dystrophy. In another aspect, the cells express premature termination codons, which cause ~10-15% of genetic diseases. In a further aspect, the animal cell is a human cell or the animal is a human patient. The following examples are intended to illustrate, and not limit the embodiments disclosed herein. Experimental Materials and Methods Preclinical studies utilizing engineered U1 and U7smOPT snRNAs have already shown promise for the inclusion and exclusion of exons to treat disease20, 21. In fact, an Atty. Dkt. No.: 114198-6910 AAV9-mediated U7smOPT snRNA gene therapy to treat boys with DMD exon 2 duplications is currently in Phase I / II clinical trials (ClinicalTrials.gov ID: NCT04240314). Based on this established track record, and the fact that most other U snRNAs are recruited downstream of U1, Applicant concentrated on these two U snRNAs. U1 snRNAs (bound by highly expressed U1A, U170K, U1C, and members of the Sm core) initiate the major spliceosome to splice introns out of pre-mRNA. Meanwhile, U7 snRNAs initiate the 3′ end processing of non-polyadenylated histone pre-mRNAs. Researchers previously mutated U7 snRNAs into U7smOPT snRNAs that bind only the Sm core, a key component of the majority of splicing U snRNAs, and not LSm proteins. With backbone sizes of 153nt and 45nt respectively, U1 and U7smOPT snRNAs are comparatively small and easily encodable in a variety of genetic delivery vehicles, from lipid nanoparticles to adeno-associated virus. A>I editing with engineered U snRNAs. Of the existing single-component A>I programmable guided RNA scaffolds, circularized ADAR-recruiting RNAs (cadRNAs) have demonstrated potent editing with a simple design10, 11. Due to their elegant circularization by autoligating twister ribozymes, cadRNAs effectively withstand degradation by exoribonucleases to sustain strong expression in cells. cadRNAs contain a C-mismatch guide with typically 100nt homology regions flanking either side of the mismatched C and occasionally mismatches and loops throughout these flanking regions to inhibit spurious bystander editing by ADAR. Due to their shared nuclear localization with ADAR, U snRNAs may be more efficient A>I editors than cadRNAs. Moreover, spliceosomal component Sm proteins have been found to associate with ADAR1 and ADAR222. To test this conjecture, Applicant replaced the cadRNA backbone (in a U6 promoter / U6 terminator cassette) with either the U7smOPT backbone (in a U7 promoter / U7 terminator cassette) or U1 snRNA backbone (in a U1 promoter / U1 terminator cassette) at the 3′ end of fixed C-mismatch guides (FIG. 1A). A head-to-head A>I editing test of the two U snRNAs against cadRNA across seven previously published endogenous loci and associated C-mismatch guide sequences in HEK293T cells revealed several findings (FIG. 1B)10. First, U1 snRNA almost invariably performed more poorly than U7smOPT snRNA. Applicant reasoned that its greater molecular complexity and proclivity for splicing machinery recruitment caused this limiting effect, and Atty. Dkt. No.: 114198-6910 so Applicant disregarded U1 snRNA as a construct for the remainder of our study. Second, although U7smOPT snRNA bested cadRNA across four of the seven loci, neither initially appeared a clear winner. Finally, U7smOPT snRNA most unambiguously outshined cadRNA editing performance at SMAD4 and FANCC, loci for genes with the highest exon counts. To test this theory further, Applicant compared U7smOPT snRNA and cadRNA performance on eight new loci of genes with progressively higher exon counts (FIG.1C). On all target genes except BLM, U7smOPT snRNA convincingly outperformed cadRNA across high exon count gene loci. Lending more credence to our theory, the ratio of U7smOPT snRNA-to-cadRNA editing efficiency over the fifteen tested genes correlates moderately and statistically significantly with exon count (Pearson correlation coefficient r = 0.6282, p = 0.0121) in contrast with unsupported alternative hypotheses of gene length (r = -0.0414, p = 0.8836) or mRNA nuclear export rate as reported in a recent study (r = 0.1436, p = 0.6096)23. Given that high exon count genes tend to be larger and more prone to accumulating disease-relevant mutations (as is the case for DMD in which ~15% of Duchenne muscular dystrophy- implicated mutations are nonsense)24, U7smOPT snRNAs present an attractive new modality for treating PTC diseases. Off-target genetic perturbations of A>I snRNAs. Next, Applicant asked how U7smOPT snRNAs compared to cadRNAs with respect to off-target genetic perturbations. Selecting one guide for which cadRNA outperformed (RAB7A targeting) and one for which U7smOPT snRNA outperformed (DMD targeting), Applicant performed differential gene expression analysis with DESeq2 on two replicates of RNA sequencing data from each condition compared to empty vector (significance cutoffs of |log2(fold change)| > 0.5 and adjusted p-value < 0.05)25. In analyzing the data, Applicant removed apparent overexpression of DMD due to a library preparation artifact as has been done in previous work (FIG. 7)10. In either case, U7smOPT snRNA produced far fewer genetic perturbations (~4-fold to 8-fold) than did cadRNA, both in genes upregulated and downregulated. Notably, more misregulated genes are shared exclusively between the two cadRNA conditions (267 genes) than between either the two DMD-targeting conditions (42 genes) or the two RAB7A conditions (121 genes) (FIG. 8). This paradox suggests that guide RNA-independent mechanisms dominate the off-target landscape. Pathway analysis with Metascape of perturbed genes conserved across both cadRNA conditions and absent from Atty. Dkt. No.: 114198-6910 either U7smOPT snRNA condition showed notable downregulation of Herpes simplex virus 1 infection and double strand break repair via synthesis strand annealing (FIG. 8)26. These results imply that structured, stable cadRNAs may be inducing an innate immune response and acting as templates for homologous recombination, either of which would be highly problematic for cells. While cadRNA may be more genetically perturbative overall, Applicant expected U7smOPT snRNA to generate more splicing changes in the transcriptome. To test this hypothesis, Applicant performed local splicing variation (LSV) analysis with MAJIQ of the RNA sequencing data sets (significance cutoff of p-value < 0.05) (FIG. 2B, FIG. 9)27. Astonishingly, for both guides across three different thresholds of differential Percent Spliced In (dPSI), cadRNA produced ~1.5x to ~2x more significant LSV events than did U7smOPT snRNA. Applicant attribute this unexpected finding not to directly guided splicing perturbations, but rather to pleiotropic effects stemming from cadRNA-mediated downregulation of splicing factors (24 vs. 7 for DMD targeting and 21 vs. 3 for RAB7A targeting, cadRNA vs. U7smOPT snRNA, respectively). Finally, Applicant examined the number of off-target A>I editing events absent from both empty vector replicates and present across cadRNA and U7smOPT replicates with our established SAILOR pipeline (significance cutoff of >75% confidence) (FIG. 2C). In the case of each guide—for both exonic and non-exonic edit sites, and at various edit fraction thresholds—U7smOPT snRNA generated consistently more transcriptome-wide A>I edits than did cadRNA. While concerning, these increased off-target edits do not appear to contribute significantly to transcriptome-wide genetic perturbations (FIG. 2A). Moreover, they could be mitigated by reducing the guide length or by introducing mutations to disrupt RNA-RNA hybridization against concerning homology-mediated off-target sites. Nuclear RNA base editing with A>I snRNAs. Applicant reasoned that the seeming contradiction between higher cadRNA-mediated genetic perturbations and U7smOPT snRNA transcriptome-wide A>I edits could be reconciled by known more durable expression of cadRNAs (and accompanying antisense knockdown of transcripts) coupled with higher localization of U7smOPT snRNAs to the ADAR protein-enriched nucleoplasm. Whereas U snRNAs spend most of their life cycle in Atty. Dkt. No.: 114198-6910 the nucleoplasm28, circular RNAs are actively exported to the cytosol29. This localization hypothesis would also explain why A>I snRNA (C-mismatch guide with U7smOPT snRNA backbone) outperforms cadRNA on high exon count gene mRNAs, which typically persist longer in the nucleus due to more extensive splicing prior to nuclear export. To test the localization hypothesis, Applicant devised a subcellular localization quantitative polymerase chain reaction (qPCR) assay whereby qPCR performed on both A>I scaffolded guides and genes from nuclear and cytosolic fractionated RNA enables inferred nuclear-to-cytosolic ratio comparison between A>I snRNA and cadRNA for equivalent guides (FIG. 3A). As expected, across three different guides A>I snRNA localized more highly to the nucleus than did cadRNA, with a sample-matched NEAT1 positive control showing no significant difference in nuclear-to-cytosolic ratio between conditions (FIG. 3A). Applicant orthogonally validated the enriched nuclear localization of A>I snRNA by single- molecule Rolling Circle Amplification Fluorescence In Situ Hybridization (RCA FISH), demonstrating that on average A>I snRNAs reside ~2 microns closer to the nucleus than cadRNAs (FIG. 3B). Additionally, RCA FISH enabled us to quantify the expression of cadRNA relative to A>I snRNA, which for the GAPDH guide is ~5-fold and likely impacts relative A>I editing performance. (Unlike the subcellular localization qPCR experiment in which cadRNA qPCR threshold cycles for nuclear and cytosolic fractions cancel each other out, cadRNA and A>I snRNA qPCR threshold cycles cannot be directly compared to each other due to differences in circularized- and linear-templated strand-displacing cDNA synthesis.) Given this exciting nuclear localization discovery, Applicant wondered whether A>I snRNAs could be leveraged to edit lncRNAs and pre-mRNAs, applications not robustly demonstrated with single-component A>I programmable guided RNA scaffold modalities. To boost editing activity in these subsequent experiments, Applicant engineered U7smOPT scaffold A>I snRNAs within a U1 promoter / U1 terminator cassette for more efficient construct expression (FIG. 10). Applicant first targeted three well characterized lncRNAs: HOTAIR, MALAT1, and XIST (FIG. 3C). As predicted, A>I snRNA outperformed cadRNA in all cases. Next, Applicant targeted pre-mRNA 3′ splice sites (3′ss), whose disruption leads to exon exclusion (FIG. 3D). In addition to testing A>I snRNA and cadRNA, Applicant included an antisense snRNA condition (C-mismatch eliminated from guide) to control for Atty. Dkt. No.: 114198-6910 the effects of spliceosomal assembly steric hindrance. Applicant first tested three 3′ss contexts for which native A>I editing has previously been implicated in splicing perturbation through ADAR knockdown: DENND4A, FBXL4, and PDE4DIP (FIG. 3E, FIG. 11)30. A>I snRNAs edited all three pre-mRNA loci more efficiently than the other conditions, with editing rates ranging from ~10-30%. These increased editing rates generally translated to improved exon skipping of A>I snRNA over both cadRNA and antisense snRNA, except for PDE4DIP in which case splicing changes fell below the sensitivity of the RT-PCR assay and a weakly editing cadRNA degraded the transcript. Finally, Applicant tested three additional 3′ss contexts for which CRISPR-Cas9 adenine deaminase base editing of DNA results in exon skipping (FIG. 3F, FIG. 12)31. Again, A>I snRNA outperformed cadRNA in editing efficiency (~20-85%) as well as exon skipping (~10-65%). These splicing results indicate a new use of A>I snRNAs whose efficacy above antisense-mediated steric hindrance of spliceosomal assembly may depend largely on cis-splicing factors. Increased pseudouridylation efficiency with U>Ψ snRNAs. Given our success in localizing A>I snRNAs to the nucleus for enhanced A>I editing, Applicant applied a similar approach to U>Ψ RNA base editing. H / ACA snoRNAs, which catalyze U>Ψ modification (editing) of rRNAs and snRNAs, localize predominantly to the nucleolus. Applicant hypothesized that more nucleoplasmic localization of programmable guided H / ACA snoRNAs via fusion to a U7smOPT snRNA backbone would direct the snoRNAs away from the nucleolus for more efficient U>Ψ editing on coding RNAs (FIG. 4A). To test whether an snRNA approach could enhance U>Ψ editing, Applicant designed a monocistronic, internally controlled dual-luminescence reporter harboring a Cystic fibrosis- implicated PTC from human CFTR (W1282X) between Renilla and Firefly luciferase (FIG. 4B). In a co-transfection experiment in HEK293T cells, expression of an established CFTR target-guided H / ACA snoRNA increased FLuc / RLuc luminescence ratio ~4x over a negative control IDUA target-guided H / ACA snoRNA, validating the assay’s sensitivity as a proxy for PTC readthrough. Of all linkers tested between CFTR target-guided H / ACA snoRNA and U7smOPT snRNA backbone, both the (c)8 and (g)8 linkers (5′-cccccccc-3′ and 5′-gggggggg- 3′) resulted in significant FLuc / RLuc luminescence ratio increases, with (g)8 linker raising Atty. Dkt. No.: 114198-6910 the luminescence ratio by ~70% above the snoRNA condition but a (g)8 tail without U7smOPT snRNA backbone having no significant effect. Pseudouridylation quantitation by BID-Seq revealed a concomitant U>Ψ editing rate increase of ~40% for (c)8 and (g)8 linker conditions (~70% editing) above the snoRNA condition (~50% editing), indicating a superlinear relationship between protein-level PTC readthrough and pseudouridylation rates (FIGS. 13A – 13C)32. Meanwhile, neither of the (a)8 and (u)8 linkers (5′-aaaaaaaa-3′ and 5′- uuuuuuuu-3′) increased the luminescence ratio. Considering these results, Applicant suspect that unlike the other linkers tested the (c)8 and (g)8 linkers help stabilize the expanded RNA scaffold. Potentially the (c)8 and (g)8 linkers, when followed by U7smOPT snRNA backbone, form an endoribonuclease-resistant secondary structure that prevents 3′-end processing of the H / ACA box snoRNA. On the other hand, the (a)8 and (u)8 linkers may recruit poly(A)-binding and poly(U)-binding proteins, respectively, that destabilize the construct. Applicant next tested the H / ACA box snoRNA-(g)8 linker-U7smOPT snRNA backbone fusions (U>Ψ snRNAs) on three endogenous loci in HEK293T cells and quantified U>Ψ editing rate using targeted amplicon CMC (N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide) sequencing (FIG. 4C, FIGS. 13D – 13E)16. On all three loci, two with statistical significance and one by over 2-fold, U>Ψ snRNAs outperformed H / ACA box snoRNAs as predicted (pseudouridylation rates of ~20-40%). This generalization suggests that increased RNA-guided targeted pseudouridylation can be achieved without cytoplasmic DKC1 overexpression, a reportedly successful strategy which nevertheless poses the risk of promoting cancer progression16. Given that U>Ψ snRNAs reproducibly enhance pseudouridylation over H / ACA box snoRNAs, Applicant sought to assess our working hypothesis of snRNA-mediated subcellular localization into the nucleoplasm and out of nucleolus. However, RCA FISH of the EEF2 guide on both U>Ψ editing RNA scaffolds did not conclusively demonstrate either increased nuclear localization or decreased nucleolar localization of the U>Ψ snRNA compared to the H / ACA box snoRNA (FIG. 14A). Notwithstanding these results, it is conceivable that snRNAs may localize the constructs to other subnuclear structures such as nuclear speckles, where DKC1 has been found to reside33. As an alternative hypothesis, U>Ψ snRNAs may achieve higher cellular abundance through Sm core-mediated stabilization and Atty. Dkt. No.: 114198-6910 thus greater construct-to-target stoichiometry. Guide-specific qPCR revealed this hypothesis to be the case for both CFTR- and ACTB-targeting constructs, but importantly for neither EEF2- nor RPS6-targeting constructs (FIG. 14B). Applicant therefore posit that the snRNA enhances pseudouridylation activity through a balance of both greater construct stability and subnuclear localization / effector recruitment. Finally, applicant tested the ability of the U>Ψ snRNA to improve nonsense-mediated mRNA decay (NMD) rescue in a human bronchial epithelial Cystic fibrosis model (FIG. 4D). PTCs lead to NMD via the downstream presence of exon junction complex(es), and PTC suppression rescues PTC-harboring mRNAs from NMD. Applicant transduced 16HBE14o- cells harboring the CFTR-W1282X mutation with lentivirus encoding empty vector, CFTR-targeting snoRNA, or previously optimized CFTR-targeting U>Ψ snRNA construct (FIG. 4A). Treatment with CFTR-targeting U>Ψ snRNA increased CFTR expression by ~2-fold when evaluated by qPCR against two distinct housekeeping genes (GAPDH and ANXA5). However, treatment with CFTR-targeting snoRNA at an equivalent transgene expression level did not achieve discernible NMD rescue. Encouragingly, this enhanced activity of U>Ψ snRNAs will enable lower dosage—and thus safer therapeutics— for the same PTC suppression efficacy. DISCUSSION RNA base editing by programmable single-component guided RNA scaffolds has demonstrated promise as both a minimally invasive and target-specific approach to gene editing. In this study, Applicant engineered U snRNAs to enhance such systems for A>I and U>Ψ editing on mammalian coding transcripts. In either base editing case, snRNAs improved system safety and / or efficacy performance over the state-of-the-art with an aspiration toward preclinical targeted suppression of premature termination codon diseases. Given that Sm core proteins are highly conserved and expressed in all mammalian cells, Applicant expect the findings to translate effectively to other cell types and broadly to PTC disease therapeutics. Excitingly, Applicant showed that U>Ψ snRNA significantly increases PTC readthrough over state-of-the-art engineered snoRNA in a human cellular disease model of Cystic fibrosis. Given the generalizability of enhanced pseudouridylation to other endogenous mRNA targets and an observed superlinear relationship between protein-level Atty. Dkt. No.: 114198-6910 PTC readthrough and pseudouridylation rates, U>Ψ snRNAs likely could benefit the development of PTC disease gene therapies broadly. Beyond genetically encoded gene therapies, U>Ψ snRNAs could also be administered to patients through non-viral delivery methods such as lipid nanoparticles. Here, the snRNA backbone may further aid in nuclear delivery, as snRNAs spend part of their life cycle at the cytoplasmic SMN complex before returning to the nucleus34. More efficient genetically encodable single-component RNA-guided editing of RNA noncoding regions, including the 3′ splice sites of pre-mRNA, opens other therapeutic opportunities. For example, RNA base editor snRNAs could edit intronic RNA-binding protein (RBP) motifs to displace destabilizing RBPs and increase nuclear RNA expression. When coupled with single-component RNA-guided translational activation systems, RNA base editor snRNAs could provide an additional boost to protein expression35. Importantly, snRNA enhancements to RNA base editing systems are guide- independent, suggesting an approach that will benefit researchers even as they orthogonally optimize RNA guides for on-target base editing efficiency and specificity36. METHODS Cloning of plasmids. Plasmids with guides / snoRNAs (guides / sno RNAs shown in Tables 1 and 2) were subcloned into pUC19 (N3041S, NEB) and pcDNA 3.1(-) (V79520, Life Technologies Corporation), both digested with EcoRI-HF (R3101T, NEB) and BamHI- HF (R3136T, NEB), by Gibson Assembly Master Mix (E2611L, NEB) with specified sequences of oligonucleotides. Similarly for lentivirus plasmids, constructs encoding reporter-validated CFTR-targeting small RNAs were ordered as gene fragments (Twist). Fragments were assembled by Gibson assembly into a lentiviral transfer vector containing an Ef1a core-eGFP-PuroR cassette (Table 1). Gibson assemblies for non-lentiviral plasmids were transformed in Mix & Go! Competent Cells-JM109 (T3005, Zymo Research Corporation) and plated on LB agar plates with antibiotic. Gibson assemblies for lentiviral plasmids were transformed in One Shot Stbl3 Chemically Competent E. coli (C737303, ThermoFisher Scientific). Colonies for all plasmids were cultured in LB media with antibiotic, then miniprepped with QIAprep Spin Miniprep Kit (27106, Qiagen). Resulting miniprepped plasmid DNA was sequenced by Primordium Labs and verified by SnapGene. Atty. Dkt. No.: 114198-6910 Cell culture. Human HEK 293T cells (632180, Takara Bio) and U-2 OS cells (HTB- 96, ATCC) were maintained in D10 (DMEM (4.5 g / L D-glucose) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin (10,000 U / mL) (Gibco) at 37°C with 5% CO2. Cells were periodically passaged once at 70-90% confluency by dissociating with TrypLE Express Enzyme (Gibco) at a ratio of 1:10. 16HBE14o- CFTR-W1282X human bronchial epithelial cells (Cystic Fibrosis Foundation) were maintained in 16HBE14o- Expansion Medium (Alpha-MEM (M2279- 500ML, Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (Gibco), 1% GlutaMax (ThermoFisher Scientific), and 1% Penicillin-Streptomycin (10,000 U / mL) (Gibco)) at 37°C with 5% CO2. Prior to plating cells, flasks were coated for ~2 hours with Fibronectin / Collagen ECM Mixture (97 mL Alpha-MEM (M2279-500ML, Sigma-Aldrich), 2mL Human Fibronectin Stock (0.5 mg / mL in Alpha-MEM) (F2006-5MG, Sigma-Aldrich), and 1mL PureCol (3 mg / mL in 0.01 N HCl) (5006-15MG, Sigma-Alrich)). Fibronectin / Collagen ECM Mixture was aspirated, but not rinsed, following coating. Cells were periodically passaged once at 90-95% confluency by dissociating with TrypLE Express at a ratio of 1:10. 16HBE14o- CFTR-W1282X cells were genotyped by harvesting in QuickExtract DNA Extraction Solution (QE09050, LGC, Biosearch Technologies), followed by PCR with primers containing sequences 5′-GGTCAGGATTGAAAGTGTGCA-3′ and 5′- CTATGAGAAAACTGCACTGGA-3′, PCR purification, and amplicon sequencing (Plasmidsaurus). Transfections. Plasmids were transfected into human HEK 293T cells under passage 30 by jetOPTIMUS DNA transfection Reagent (76299-632, VWR International). For A>I RNA extractions, cells were plated in 48-well tissue culture plates and transfected at ~60% confluency with 250 ng of plasmid DNA. For U>Ψ RNA extractions, cells were plated in 12- well tissue culture plates and transfected at ~60% confluency with 1 ug of plasmid DNA. For luciferase reporter assay, cells were plated in 96-well tissue c ture plates and transfected at ~60% confluency with 100 ng of plasmid DNA (75ng of guide plasmid DNA, 25 ng of reporter plasmid DNA). Under these conditions, a transfection efficiency of 80+% was achieved routinely. Atty. Dkt. No.: 114198-6910 RNA extraction, A>I editing quantification, and RNA sequencing library preparation. At a time 48 hours after transfection, cells were washed twice with PBS, then RNA was extracted by RNeasy Plus Mini Kit (74136, Qiagen) with an elution volume of 30 uL. For A>I editing quantification, cDNA synthesis was carried out by ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB) with 3 uL RNA in a 10 uL total volume using oligo(dT) primers supplied with the kit. PCR with 500nM specified primers (Table 3) was carried out by NEBNext Ultra II Q5 Master Mix (M0544L, NEB) with Tm of 68°C and 30 second extension time for 30 cycles for mRNA products and for 32 cycles for pre-mRNA products. PCR products were purified by QIAquick PCR Purification Kit (28106, Qiagen) and submitted to Sanger sequencing to quantify A>I editing. RNA sequencing library preparation was carried out by Illumina Stranded mRNA Prep, Ligation (20040532, Illumina) with 1 ug RNA using manufacturer’s standard protocol. The RNA sequencing library was sequenced on an Illumina NovaSeq X Plus 10B under the PE100 configuration with a target sequencing depth of ~50 million reads per sample. Correlation modeling of U7smOPT snRNA vs. cadRNA A>I editing. Analysis was performed on the 15 genes targeted in FIG. 1B and 1C. U7smOPT snRNA-to-cadRNA performance ratios were calculated as the ratios of the mean editing efficiencies for each respective target. Exon counts were taken from GRCh38.p14. Gene lengths were calculated as the difference between start and end base indices of genes from GENCODE v44 (GRCh38.p14). Mean nuclear export rates of gene mRNAs were calculated as the average of 2 replicates of k_nucexp_from_nucres.Mean in K562 cells from Table S1of Ietswaart et al., 2024. Pearson’s linear correlation coefficients were taken of performance ratio vs. each hypothesis (exon count, gene length, mean nuclear export rate) across all targets. Data were analyzed and plotted in MATLAB (v R2024a). RNA sequencing alignment. Reads were checked for quality and adapter sequences using FastQC. Paired reads were then aligned to the genome using STAR aligner version 2.7.6a. A genome index for alignment was generated using the GENCODE v.44 hg38 primary assembly with the following command line parameters: --genomeFastaFiles GRCh38.primary_assembly.genome.fa, --sjdbGTFfile gencode.v44.primary_assembly.annotation.gtf, and --sjdbOverhang 100. Paired FASTQ files were then mapped to the genome with default options and --outSAMtype BAM unsorted. Atty. Dkt. No.: 114198-6910 Aligned reads in BAM format were sorted by position using samtools sort (version 1.3.1) with default parameters. Sorted BAM files were indexed using samtools index with default parameters. Differential gene expression analysis. Differential expression of aligned RNASeq reads relative to controls was analyzed using subreadfeaturecounts version 1.5.3 followed by DESeq2 version 1.39.3. Gene counts for control (pUC19) and experimental replicates were collected in count matrices using subreadfeaturecounts (v 1.5.3). Gene count matrices for each condition were generated using the function subreadfeaturecounts::featureCounts with the following parameters: -p (for paired-end reads), -a gencode.v44.primary_assembly.annotation.gtf, -t exon, -g gene_name, --primary (to count only the primary alignment for multimapping reads) -Q 255 (the minimum quality score that a counted read must satisfy), and --ignoreDup (to exclude duplicate reads). Matrices were then loaded into a jupyter R notebook as a ‘counts’ object using R::read.table, excluding the first 5 lines which do not contain counts. Counts in the R data frames were labelled by condition followed by labels.df <- DataFrame(condition = labels, row.names = colnames(counts)). Count matrices were converted into DESeq2 datasets with DESeq2:: DESeqDataSetFromMatrix. Genes with no expression across samples were removed. Differentially expressed genes were then identified using DESeq2::DeSeq with default parameters. Results were collected and saved as .csv files using DeSeq2:: results and R:: write.table. Volcano plots of significantly up and downregulated genes for each condition were generated using RNAlysis 2 (v 3.9.2). Genes from DESeq2 (v 1.39.3) outputs were plotted using DESeqFilter.volcano_plot() with alpha (p-adj significance threshold) set to 0.05 and Log2FC thresholds of 0.5 and 1.00. An additional counts matrix containing gene counts for all experimental and control replicates was used to generate PCA plots with RNAlysis 2 (v 3.9.2). Counts were normalized by relative log expression with RNAlysis2::CountFilter.normalize_rle(). Genes with no expression across columns were filtered from analysis using CountFilter.filter_low_reads() with a threshold of 0. PCA was performed using Atty. Dkt. No.: 114198-6910 RNAlysis2::CountFilter.pca() function with default parameters, except for power_transform = False. GSEA (v 4.3.2) was used for pathway analysis of the ranked and filtered gene lists. DESeq2 output tables were copied into Microsoft Excel (v 16.66.1). Ranking metrics for each gene were calculated by the formula =SUM(SIGN([@log2FoldChange])*(- LOG10([@pvalue]))). Genes were then filtered by a LogFC threshold of + / - 0.5. Ranked lists containing gene names and ranking metrics were copied into .rnk files. For DMD targeting guide RNAs, DMD was excluded from lists due to a library preparation artifact. Ranked lists were loaded into GSEA and analyzed using GSEAPreRanked with the following parameters changed from default: gene set = c5.go.bp.v2023.2.Hs.symbols.gmt, collapse = no_collapse, Gene pathway analysis. Significantly upregulated and downregulated genes identified in both cadRNA datasets but not in either U7smOPT snRNA dataset were analyzed for gene pathway signficance with Metascape (v3.5.20240101) using as background all genes without an “NA” p-adj significance in all replicates of cadRNA and U7smOPT snRNA datasets from the DESeq2 pipeline. Differential splicing analysis. MAJIQ and VOILA software packages (v 2.5) were used to assess splicing variation between treatment groups (cadRNA and U7 smOPT) and pUC19 controls. MAJIQ builder constructed splice graphs, and MAJIQ quantifier was used to quantify delta percent spliced in (dPSI) of local splicing variations (LSVs) under default conditions at known splice sites. VOILA TSV function was used select only genes with at least one LSV with 95% > probability the dPSI > x ( P(|dPSI| > x) > 0.95). Plotting LSVs was performed with PRISM and R. Splicing factor analysis. Significantly perturbed splicing factors were identified as a subset of significantly upregulated and downregulated genes from both cadRNA and U7 smOPT datasets contained within GOBP_RNA_SPLICING (GO:0008380) at the Gene Ontology Consortium Transcriptome-wide A>I editing analysis. Transcriptome wide RNA editing was quantified with SAILOR (v 1.1.0). Base quality MD tags for aligned reads were generated using samtools (v 1.3.1) calmd with -b (for bam files) and the GENCODE v. 44 GRCh38.primary_assembly.genome.fa sequence file. Reads containing MD tags were then Atty. Dkt. No.: 114198-6910 analyzed with the SAILOR (v 1.1.0) cwl workflow. The reference genome used was the same used for alignment and the addition of MD tags. Edit sites were only considered significant if their SAILOR confidence level was greater than 0.75. Edit sites were identified as exonic if they intersected by bedtools intersect command (parameters “-s -wa -a”) with features labeled as “exon” from gencode.v44.primary_assembly.annotation.gtf. All other edit sites were identified as non-exonic. Edit fraction thresholds (1 = 100%) were applied on SAILOR output POST_PSEUDOCOUNT_EDIT%. Subcellular localization qPCR. Cells were washed twice with ice cold PBS, then spun down for 5 minutes at 300 g, with supernatant aspirated. Cells were then resuspended completely by gentle pipetting with 150 uL Buffer A (15mM Tris-HCl pH 8, 15mM NaCl, 60mM KCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 0.5mM spermidine, 10U / mL SUPERase•In RNase Inhibitor (AM2694, ThermoFisher Scientific)). To this solution was added and mixed by inversion 150 uL of 2x lysis buffer (Buffer A with 0.5% NP-40). Mixture was incubated for 8 minutes at 4°C, then spun down for 5 minutes at 400 g. The top 200 uL of supernatant was carefully removed and placed into a new tube (this is the cytosolic fraction). The remaining supernatant was removed and discarded from the nuclear pellet, and this pellet was resuspended in 1mL of RLN buffer (50mM Tris-HCl pH 8, 140mM NaCl, 1.5mM MgCl2, 0.5% NP-40, 10mM EDTA pH 8, 10U / mL SUPERase•In RNase Inhibitor (AM2694, ThermoFisher Scientific)). This nuclear resuspension was incubated for 5 minutes at 4°C. During the incubation, the cytosolic fraction was spun again for 1 minute at 500 g, and its supernatant was collected into a new tube. 500 uL Trizol LS (10296010, Invitrogen) was added to this cytosolic fraction. The nuclear fraction was spun down once more for 5 minutes at 500 g. Supernatant was removed from the nuclear fraction pellet, and 500 uL TRIzol (15596018, Invitrogen) was added to the nuclear fraction pellet. To both TRIzol homogenizations was added 1 uL of GlycoBlue Coprecipitant (AM9516, ThermoFisher Scientific). Then RNA was extracted by phenol-chloroform extraction, followed by ethanol precipitation. cDNA synthesis was carried out by ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB) with 6 uL RNA in a 20 uL total volume using random hexamer primers supplied with the kit. Prior to qPCR, nuclear fraction cDNA was diluted 1:2 with nuclease- free water, and cytosolic fraction cDNA was diluted 1:60 with nuclease-free water. qPCR Atty. Dkt. No.: 114198-6910 with 500nM specified primers (Supplementary Table 4) was carried out by PowerTrack SYBR Green Master Mix (A46109, ThermoFisher Scientific) using the CFX Opus 384 (Bio- Rad) and qPCR parameters of 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60C°C for 1 minute. From the qPCR Cq values, subcellular (nuclear and cytosolic) guide and NEAT1 expression levels were normalized relative to their subcellular GAPDH expression controls, and then the ratio of these normalized subcellular expressions was calculated. Data were analyzed and plotted in MATLAB (v R2024a). Splicing isoform quantification. For splicing isoform quantification, cDNA synthesis was carried out by ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB) with 3 uL RNA in a 10 uL total volume using oligo(dT) primers supplied with the kit. PCR with 500nM specified primers (Supplementary Table 4) was carried out by NEBNext Ultra II Q5 Master Mix (M0544L, NEB) with Tm of 68°C and 30 second extension time for 32 cycles. PCR products were purified by QIAquick PCR Purification Kit (28106, Qiagen), with 50% of eluted volume run on a E-Gel EX Agarose Gels, 2% (G402022, ThermoFisher Scientific) for ~15 minutes with E-Gel Ultra Low Range DNA Ladder (10488096, ThermoFisher Scientific) and E-Gel 50 bp DNA Ladder (10488099, ThermoFisher Scientific). Gels were visualized using the Azure Biosystems c600. Gel bands were quantitated using GelAnalyzer (v19.1), with Percent Spliced In (PSI) values calculated after adjusting bands for relative molecular weights. Luciferase reporter assay. At a time 48 hours after transfection, cell media was changed, and luminescence was generated using the Dual-Glo Luciferase Assay System (E2920, Promega) according to manufacturer’s standard protocol. Luminescence was measured using a Tekan infinite 200Pro plate reader with the Costar 96 flat white setting, automatic attenuation, and integration times of 500ms for Firefly and 100ms for Renilla. Data were analyzed and plotted in MATLAB (v R2024a). In vitro transcription of pseudouridine standards. Template DNA is first generated by a two-stage PCR on a DNA oligo containing respective target sequence context (Supplementary Table 4). First, the oligo is PCR amplified for two cycles with pseudouridylation standard primers (PCR1). This PCR amplicon was then used as a template for a 15-cycle reaction with (PCR2). The resulting PCR amplicon was PAGE purified and Atty. Dkt. No.: 114198-6910 divided into two T7 in vitro transcription reactions (MEGAshortscript, Invitrogen), containing either standard triphosphate pool (for unmodified standards), or with substitution of UTP for ΨTP (for pseudouridine standards). After a 120-minute in-vitro transcription, the reaction is DNase treated (TURBO DNase, Invitrogen), and RNA is PAGE purified before use in CMC or BID-Seq reactions. Targeted bisulfite sequencing (BID-Seq). DNaseI-treated total RNA was prepared for BID-Seq as described previously (Dai et al., 2022). Specifically, 8.5 μL of RNA was combined with 45 μL 2.4M Na2SO3, 0.36M NaHSO3, and heated at 70°C for 3 hours. The reaction was then combined with 75 μl RNase-free H2O, 270 μl RNA binding buffer (Zymo RNA Clean and Concentrator), and 400 μl ethanol and loaded onto a Zymo RNA Clean and Concentrator-5 column. The column was washed 1x with 200 μL RNA wash buffer (Zymo), then loaded with 200 μL RNA desulfonation buffer (Zymo) and incubated at room temperature for 90 minutes. Then, desulfonation buffer is cleared by centrifugation and the column was washed 2x with 700 RNA wash buffer (Zymo). Bisulfite-treated RNA is eluted in 10.5 μL nuclease-free H2O. For reverse-transcription, random-hexamer primers were annealed to RNA by combining 5 μL RNA, 1 μL 50 mM random-hexamers, 10 mM dNTPs, 6 μL water and heating at 65°C for 5 minutes, then snap-chilling on ice for 1 minute. Primer- annealed RNA was combined with 4 μL 5x SSIV buffer, 100 mM DTT, 1 μL RNasein Plus, 1 μL SSIV, and incubated at 23°C for 10 minutes, 55°C 10 minutes, 80°C 10 minutes. RNA was hydrolyzed by addition of 5 μL 1M NaOH and heating at 95°C for 5 minutes, then quenching with 5 μL 1M HCl. cDNA was then purified with MyOne Silane Dynabeads (Invitrogen) and eluted in 20 μL nuclease-free H2O. Gene-specific primers (Supplementary Table 4) were used to PCR-amplify the respective target site, and PCR products were purified using Ampure-XP beads (Beckman Coulter) and pooled at equal concentration before submission for amplicon sequencing (Genewiz). Raw FASTQ files were aligned to target reference sequences using bbmap, and the resulting bam files sorted and indexed using samtools. Deletion rate at each position was quantified using the samtools mpileup command and custom python scripts. Effective pseudouridylation rates were calculated by normalizing to the 100% ΨTP CFTR synthetic RNA standard. Data were analyzed and plotted in MATLAB (v R2024a). Atty. Dkt. No.: 114198-6910 Targeted amplicon CMC sequencing. To disrupt the RNA secondary structure, 5 ug of RNA per sample in ~10 uL nuclease-free water was incubated at 80°C for 5 minutes and quickly chilled on ice. Next, the denatured RNA was transferred into 100 uL of freshly prepared and sterile-filtered BEU buffer (50 mM Bicine, pH 8.5, 4 mM EDTA, 7 M urea) with 0.2 M CMC (C106402-1G, Sigma Aldrich) and incubated at 37°C for 20 min, followed by purification with ethanol precipitation. The RNA pellets were then dissolved in 50 uL Na2CO3buffer (50 mM Na2CO3pH 10.4, 2 mM EDTA) and incubated at 37°C for 2 hours, followed by ethanol precipitation. Next, the RNA pellets were dissolved in 10 uL nuclease- free water. 2 uL of random hexamer primers from SuperScript First-Strand Synthesis System for RT-PCR (11904018, ThermoFisher Scientific) were added, and the mixtures were denatured at 65°C for 5 min followed by chilling on ice. Next, 8 uL freshly prepared 2.5x reverse transcription buffer (125 mM Tris pH 8.0, 15 mM MnCl2, 187.5 mM KCl, 1.25 mM dNTPs, 25 mM DTT) was added to the RNA-primer mixtures and the mixture was incubated at 25°C for 2 min. Then 1 uL SuperScript II reverse transcriptase from SuperScript First-Strand Synthesis System for RT-PCR (11904018, ThermoFisher Scientific) was added, and the reactions were carried out at 25°C for 10 minutes, 42°C for 3 hours and 70°C for 15 minutes. For library preparation, PCR was carried out in two steps. In the first step, PCR with 500nM specified locus-specific primers (Supplementary Table 4) was carried out with 1 uL of cDNA by NEBNext Ultra II Q5 Master Mix (M0544L, NEB) in a total reaction volume of 10 uL with Tm of 65°C and 30 second extension time for 15 cycles. Then first round PCR products were cleaned up by 1.8x AMPure XP beads (A63881, Beckman Coulter) and eluted in 11 uL. In the second step, PCR with 500nM specified NGS universal and barcoded primers (Table 3) was carried out with 10 uL of cleaned up first round PCR product by NEBNext Ultra II Q5 Master Mix (M0544L, NEB) in a total reaction volume of 20 uL with Tm of 65°C and 30 second extension time for 15 cycles. Second round PCR products were pooled and purified by QIAquick PCR Purification Kit (28106, Qiagen), then this purified pool was run on a E-Gel EX Agarose Gels, 2% (G402022, ThermoFisher Scientific) for ~15 minutes. The upper band on the gel (~250bp) was extracted and purified by QIAquick Gel Extraction Kit (28704, Qiagen) before sequencing. The targeted amplicon sequencing library was sequenced Atty. Dkt. No.: 114198-6910 on an Illumina NovaSeq X Plus 10B under the PE150 configuration with a target sequencing depth of ~5 million reads per sample. Targeted amplicon sequencing FASTQ files were collapsed along UMI (Unique Molecular Identifier) by selecting as sequence for a given 10-mer UMI in a read its first occurrence in each FASTQ file. Then only sequences with perfect 8-mer matches both upstream and downstream of target NΨN at the locus were considered. Deletion rates were calculated as the fraction of total sequences at a locus with 2 nucleotides between the perfect 8-mer matches. Mutation rates were calculated as the fraction of total sequences at a locus with 3 nucleotides between the perfect 8-mer matches and which did not have a T at the Ψ position in target NΨN. Effective pseudouridylation rates were calculated by normalizing to the 100% ΨTP ACTB, EEF2, and RPS6 synthetic RNA standards. Data were analyzed and plotted in MATLAB (v R2024a). Guide-specific qPCR to quantitate construct expression. At a time 48 hours after transfection, HEK293T cells were washed twice with PBS, then RNA was extracted by RNeasy Plus Mini Kit (74136, Qiagen) with an elution volume of 30 uL. cDNA synthesis was carried out by ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB) with 3 uL RNA in a 10 uL total volume using two different reactions per replicate: random hexamer primers supplied with the kit for GAPDH quantitation, and the primer used as reverse qPCR primer for guide quantification (Supplementary Table 4. Prior to qPCR, cDNA was diluted 1:3 with nuclease-free water. qPCR with 500nM specified primers (Table 3) was carried out by PowerUp SYBR Green Master Mix (A25742, ThermoFisher Scientific) using the CFX96 (Bio-Rad) and qPCR parameters of 50°C for 2 minutes, 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60C°C for 1 minute. From the qPCR Cq values, guide expression levels were normalized relative to GAPDH. Data were analyzed and plotted in MATLAB (v R2024a). Rolling circle amplification fluorescence in situ hybridization (RCA FISH) experiment. U-2 OS cells under passage 20 were seeded into 96-well glass-bottom microplates (Greiner Bio-One SensoPlate, cat. no. 07-000-109). Plasmids were transfected into cells at ~30% confluency by jetOPTIMUS DNA transfection Reagent (76299-632, VWR International). At three bioreplicate wells per condition, cells were transfected with 12.5 ng of Atty. Dkt. No.: 114198-6910 plasmid DNA for A>I RNA RCA FISH and 25 ng of plasmid DNA for U>Ψ RNA RCA FISH. In either case, cells were then cultured until approximately 80% confluency. Prior to fixation, cells were incubated at 65°C for 5 min, followed by rapid cooling on ice. Cells were fixed for 30 min at room temperature (RT) in a solution of 4% (wt / vol) paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15714) and 0.007% (vol / vol) glutaraldehyde (Electron Microscopy Sciences, cat. no. 16120) in 1× phosphate- buffered saline (PBS; Ambion, cat. no. AM9625). Following three washes with 1× PBS, cells were permeabilized with 0.5% (vol / vol) Triton X-100 (Lab stock) in 1× PBS for 10 min at RT. Permeabilization solution was removed by three washes with PBS containing 0.05% (vol / vol) Tween-20 (PBS-T; VWR, cat. no. 100216-360). Target sn / snoRNAs were reverse transcribed in situ. Per well (50 uL total volume), the reaction mixture contained 1× SuperScript IV buffer (Lifetech, cat no. 18090050), 500 uM each dNTP (New England Biolabs, cat. no. N0447L), 1 uM RT primer (IDT), 0.2 mg / mL bovine serum albumin (BSA; New England Biolabs, cat. no. B9200S), 0.8 U / uL RNase inhibitor (M0314L or Thermo Fisher Scientific, cat. no. EO0384), and 20 U / uL SuperScript IV Reverse Transcriptase (Lifetech, cat no. 18090050) in RNase-free water. Plates were sealed and incubated overnight (approx. 16–18 h) at 37°C. Following reverse transcription, cells were washed three times with PBS-T and post- fixed for 30 min at RT in 1× PBS containing 3% (wt / vol) PFA and 0.1% (vol / vol) glutaraldehyde. Cells were then washed five times with PBS-T. Padlock probe ligation was performed in a 50 uL reaction volume per well containing 1× Ampligase buffer (Lucigen, cat. no. A3210K), 100 nM padlock probe (IDT, HPLC purified), 0.2 mg / mL BSA, 0.4 U / uL RNase H (Enzymatics, cat. no. Y9220L), and 0.5 U / uL Ampligase (Lucigen, cat. no. A3210K). The reaction was incubated at 37°C for 30 min, followed by 45°C for 45 min on a thermocycler with a heated lid. Cells were subsequently washed three times with PBS-T. The ligated padlock probes were amplified via RCA. The reaction mix (50 uL per well) was prepared on ice and contained 1× Phi29 buffer (Thermo Fisher Scientific, cat. no. EP0091), 5% (vol / vol) glycerol (Sigma-Aldrich, cat. no. G5516), 250 uM each dNTP, 0.2 mg / mL BSA, and 1 U / uL Phi29 DNA polymerase (Thermo Fisher Scientific, cat. no. Atty. Dkt. No.: 114198-6910 EP0091), added last to the chilled mixture. Plates were sealed, ensuring adjacent empty wells contained water to maintain humidity, and incubated overnight (approximately 18 h) at 30°C. After RCA, cells were washed three times with PBS-T. RCA products were detected by hybridization with fluorescently labeled hybridization probes (IDT, HPLC purified). Hybridization mix (100 uL per well) contained 1 uM hybridization probe in hybridization buffer (2× saline-sodium citrate (SSC; Ambion, cat. no. AM9763), 10% (vol / vol) formamide). Hybridization was performed for 30 min at RT, followed by three washes with PBS-T. Nucleoli were stained with 1 uM Nucleolar Red stain in 1× PBS for 5 min. Nuclei were counterstained with DAPI. Fluorescence imaging was performed using a Squid microscopy system (Cephalogics Inc.) with penta band filter set, 405 / 470 / 550 / 640 / 730 nm laser lines and an IMX571 camera. Objective used is Olympus 10x / 0.8 Plan Apo. Excitation wavelengths of 405 nm (DAPI), 561 nm (Nucleolar Red), and 638 nm (TYE665) were used. For A>I RNA RCA FISH, 9 images (~10-20K cells total) were taken per well. For U>Ψ RNA RCA FISH, 13 images (~20-40K cells total) were taken per well. RCA FISH analysis. RCA FISH images were analyzed in CellProfiler (v 4.2.8) with a custom project pipeline. Briefly, nuclei were identified in the 405 filter images by IdentifyPrimaryObjects with a diameter between 10- and 40-pixel units, global threshold strategy, minimum cross-entropy thresholding method, threshold smoothing scale of 1.3488, threshold correction factor of 1.0, and lower and upper bounds on threshold of 0.2 and 1.0. Clumped objects were distinguished by shape and divided by intensity. “Cell boundaries” were then identified with ExpandOrShinkObjects by an expansion of 20 pixels. RCA rolonies were first identified in the 640 filter images by IdentifyPrimaryObjects with a diameter between 1- and 10-pixel units, global threshold strategy, minimum cross- entropy thresholding method, threshold smoothing scale of 1.3488, threshold correction factor of 1.0, and lower and upper bounds on threshold of 0.07 and 1.0. Clumped objects were distinguished by shape and divided by intensity. Only RCA rolonies within “cell boundaries” (filtered by RelateObjects) were counted. For U>Ψ RNA RCA FISH images exclusively, nucleoli were first identified in 550 filter images by IdentifyPrimaryObjects with a diameter between 1- and 7-pixel units Atty. Dkt. No.: 114198-6910 (without discarding objects outside the diameter range but with discarding objects touching the border), adaptive threshold strategy, robust background thresholding method, lower and upper outlier fractions of 0.05, mean averaging method, standard deviation variance method, 1.5 # of deviations, threshold smoothing scale of 0.674, threshold correction factor of 1, lower and upper bounds on threshold of 0.02 and 1.0, and size of adaptive window of 7. Clumped objects were distinguished by intensity and divided by intensity. Only nucleoli within nuclei (filtered by RelateObjects) were counted. To calculate edge-to-edge distances between RCA rolonies and nuclei / nucleoli, nuclei / nucleoli were first converted to binary (black & white images) by ConvertObjectsToImage. Then these nuclei / nucleoli images were inverted by ImageMath using the invert operation, multiplying the first image by 1.0, raising the power of the result by 1.0, and multiplying the result by 1.0. Values less than 0 were set to 0, greater than 1 set to 1, and invalid values were replaced with 0. Morph was performed on the resulting inverted nuclei / nucleoli images with the distance operation. Distances between RCA rolonies and nuclei / nucleoli were calculated by MeasureObjectIntensity with morphed distances and images and cellular RCA rolonies as objects. Finally, edge-to-edge distances between RCA rolonies and nuclei / nucleoli were taken as the Intensity_MinIntensityEdge values from these morphed distances (values assigned as 0 if RCA rolonies were inside nuclei / nucleoli). Data were analyzed and plotted in MATLAB (v R2024a). Cystic fibrosis disease modeling. For lentivirus production, 8e6 LentiX cells (Takara) were seeded 24 hours prior to transfection into a 10 cm dish. Virus particles were packaged by standard techniques using three-plasmid transfection with VirusGen transfection reagent (Mirus). Viral supernatants were collected 72 hours post transfection, filtered using a 0.45 PVDF filter, and concentrated by peg precipitation (LentiX concentrator) then snap frozen in liquid nitrogen and stored at -80°C. Virus was functionally titered on HEK293T cells via flow cytometry with gating on eGFP. 200K 16HBE14o- CFTR-W1282X cells were seeded on ECM coated 48 well plates 24 hours before transduction. Cells were then spin- transduced with PBS containing titer adjusted concentrated virus in the presence of 5 μg / ml polybrene at 1000 RCF for 15 minutes at 37°C. Media was changed 48 hours post transduction. Cells were split every 3-5 days and cultures were expanded to a 10 cm dish over approximately three weeks. To prevent the expansion of non-transduced cells, cells were Atty. Dkt. No.: 114198-6910 cultured in 1.5 ug / mL of puromycin from days 7-14. Cells were washed twice with PBS, scraped, pelleted, and snap frozen. RNA was extracted from cell pellets by RNeasy Plus Mini Kit (74136, Qiagen) with an elution volume of 30 uL. cDNA synthesis was carried out by ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB) with 6 uL RNA in a 20 uL total volume using oligo(dT) primers supplied with the kit. qPCR with 500nM specified primers (Supplementary Table 4) was carried out by PowerTrack SYBR Green Master Mix (A46109, ThermoFisher Scientific) using the CFX Opus 384 (Bio-Rad) and qPCR parameters of 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60C°C for 1 minute. From the qPCR Cq values, CFTR and PuroR expression levels were normalized relative to GAPDH and ANXA5 housekeeping genes. Data were analyzed and plotted in MATLAB (v R2024a). Table 1: Polynucleotide Sequences Containing H / ACA box snoRNA backbone sequences with possible promoter / termination (terminator) sequences. In general, nucleotides in uppercase are nucleotides that are not part of the promoter or terminator (termination) sequences improve of the construct. For the U7smOPT sequence, which contains three uppercase letters corresponding to mutations used to render the U7 snRNA sequence into the U7smOPT snRNA sequence. For the CFTR PTC dual luciferase reporter sequence, for which uppercase and lowercase sequences alternate to indicate breaks in different regions of the plasmid. <guide> indicates where one of the one or more guide RNAs can be linked to the backbone.

[0002] Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Table 2: Exemplary Guide and snoRNA sequences. Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Table 3: Primers for PCR, qPCR, Sanger sequencing, and NGS. Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Atty. Dkt. No.: 114198-6910 Table 4 – Exemplary U snRNA Sequences Atty. Dkt. No.: 114198-6910 Annotated Polynucleotides SEQ ID NO: 126 U6 Promoter-Terminator-for snoRNAs Backbone LOCUS U6_promoter-term 258 bp DNA linear Atty. Dkt. No.: 114198-6910 Accessed 03-JUN-2025 DEFINITION natural linear DNA. ACCESSION . VERSION . KEYWORDS . SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 258) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..258 / mol_type="genomic DNA" / organism="unspecified" promoter 1..241 / label=U6 promoter / note="RNA polymerase III promoter for human U6 snRNA" misc_feature 242 / label=+1 G gap 243..252 / estimated_length=10 gap 243..252 / label=snoRNA sequence / estimated_length=10 misc_feature 253..258 / label=U6 terminator ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgnnnnnnnn nntttttt SEQ ID NO: 127 U7 Promoter-Terminator -U7smOPT_snRNA Backbone LOCUS U7_promoter-term 439 bp DNA linear Accessed on 03-JUN-2025 DEFINITION natural linear DNA. ACCESSION . VERSION . KEYWORDS . SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 439) AUTHORS MIT Atty. Dkt. No.: 114198-6910 TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..439 / mol_type="genomic DNA" / organism="unspecified" source 1..268 / chromosome="12" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" source 279..439 / chromosome="12" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" misc_feature 1..267 / label=U7 promoter / label=Goyenvalle et al. promoter misc_feature 268 / label=+1 C gap 269..278 / label=guide sequence / estimated_length=10 gene 279..323 / gene="RNU7-1" / gene_synonym="RNU7; U7.1" / label=RNU7-1 / note="RNA, U7 small nuclear 1; Derived by automated computational analysis using gene prediction method: BestRefSeq." / db_xref="GeneID:100147744" / db_xref="HGNC:HGNC:34033" / db_xref="MIM:617876" ncRNA 279..323 / gene="RNU7-1" / gene_synonym="RNU7; U7.1" / product="RNA, U7 small nuclear 1" / label=snRNA / note="Derived by automated computational analysis usinggene prediction method: BestRefSeq." / note=" / transcript_id=NR_023317.1" / db_xref="GeneID:100147744" / db_xref="HGNC:HGNC:34033" Atty. Dkt. No.: 114198-6910 misc_feature 281..291 / label=Sm binding site OPT misc_feature 324..439 / label=U7 terminator ORIGIN 1 ggcttaacaa caacgaaggg gctgtgactg gctgctttct caaccaatca gcaccgaact 61 catttgcatg ggctgagaac aaatgttcgc gaactctaga aatgaatgac ttaagtaagt 121 tccttagaat attatttttc ctactgaaag ttaccacatg cgtcgttgtt tatacagtaa 181 taggaacaag aaaaaagtca cctaagctca ccctcatcaa ttgtggagtt cctttatatc 241 ccatcttctc tccaaacaca tacgcagcnn nnnnnnnnag aatttttgga gtaggctttc 301 tggcttttta ccggaaagcc cctcttatga tgtttgttgc caatgataga ttgttttcac 361 tgtgcaaaaa ttatgggtag ttttggtggt cttgatgcag ttgtaagctt ggggtatgaa 421 ggtttgggcc acgcctggg SEQ ID NO: 128 U7 Promoter-Terminator-for UPsi snRNA Backbone LOCUS U7_promoter-term 394 bp DNA linear Accessed on 03-JUN-2025 DEFINITION natural linear DNA. ACCESSION . VERSION . KEYWORDS . SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 394) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..394 / mol_type="genomic DNA" / organism="unspecified" source 1..268 / chromosome="12" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" source 279..394 / chromosome="12" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" misc_feature 1..267 / label=U7 promoter / label=Goyenvalle et al. promoter misc_feature 268 / label=+1 C Atty. Dkt. No.: 114198-6910 gap 269..278 / label=U>Psi snRNA sequence / estimated_length=10 misc_feature 279..394 / label=U7 terminator ORIGIN 1 ggcttaacaa caacgaaggg gctgtgactg gctgctttct caaccaatca gcaccgaact 61 catttgcatg ggctgagaac aaatgttcgc gaactctaga aatgaatgac ttaagtaagt 121 tccttagaat attatttttc ctactgaaag ttaccacatg cgtcgttgtt tatacagtaa 181 taggaacaag aaaaaagtca cctaagctca ccctcatcaa ttgtggagtt cctttatatc 241 ccatcttctc tccaaacaca tacgcagcnn nnnnnnnnct tatgatgttt gttgccaatg 301 atagattgtt ttcactgtgc aaaaattatg ggtagttttg gtggtcttga tgcagttgta 361 agcttggggt atgaaggttt gggccacgcc tggg SEQ ID NO: 129 U6 Promoter-Terminator-cadRNA Backbone LOCUS U6_promoter-term 443 bp DNA linear Accessed on 05-JUN-2025 DEFINITION natural linear DNA. ACCESSION . VERSION . KEYWORDS . SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 443) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..443 / mol_type="genomic DNA" / organism="unspecified" promoter 1..241 / label=U6 promoter / note="RNA polymerase III promoter for human U6 snRNA" misc_feature 250..327 / label=required for cadRNA / label=required misc_feature 328..336 / label=A linker gap 337..346 / estimated_length=10 gap 337..346 / label=guide sequence / estimated_length=10 misc_feature 347..356 / label=A linker Atty. Dkt. No.: 114198-6910 misc_feature 357..437 / label=required for cadRNA / label=required misc_feature 438..443 / label=U6 terminator ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ccatcagtcg ccggtcccaa gcccggataa aatgggaggg ggcgggaaac 301 cgcctaacca tgccgactga tggcagaaaa aaaaaannnn nnnnnnaaaa aaaaaactgc 361 catcagtcgg cgtggactgt agaacactgc caatgccggt cccaagcccg gataaaagtg 421 gagggtacag tccacgcttt ttt SEQ ID NO: 130 U1 Promoter-Terminaor-U7smOPT_snRNA Backbone LOCUS U1_promoter-term 493 bp DNA linear Accessed on 05-JUN-2025 DEFINITION synthetic linear DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 493) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..493 / chromosome="1" / map="1p36.1" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" source join(404..413,414..458) / mol_type="genomic DNA" / organism="unspecified" source 414..458 / chromosome="12" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" misc_feature 1..402 / label=U1 promoter Atty. Dkt. No.: 114198-6910 misc_feature 403 / label=+1 A gap 404..413 / label=guide sequence / estimated_length=10 gene 414..458 / gene="RNU7-1" / gene_synonym="RNU7; U7.1" / label=RNU7-1 / note="RNA, U7 small nuclear 1; Derived by automated computational analysis using gene prediction method: BestRefSeq." / db_xref="GeneID:100147744" / db_xref="HGNC:HGNC:34033" / db_xref="MIM:617876" ncRNA 414..458 / gene="RNU7-1" / gene_synonym="RNU7; U7.1" / product="RNA, U7 small nuclear 1" / label=snRNA / note="Derived by automated computational analysis usinggene prediction method: BestRefSeq." / note=" / transcript_id=NR_023317.1" / db_xref="GeneID:100147744" / db_xref="HGNC:HGNC:34033" / db_xref="MIM:617876" / ncRNA_class="snRNA" misc_feature 416..426 / label=Sm binding site OPT misc_feature 459..493 / label=U1 terminator ORIGIN 1 taacacaggc taaggaccag cttctttggg agagaacaga cgcaggggcg ggagggaaaa 61 agggagaggc agacgtcact tccccttggc ggctctggca gcagattggt cggttgagtg 121 gcagaaaggc agacggggac tgggcaaggc actgtcggtg acatcacgga cagggcgact 181 tctatgtaga tgaggcagcg cagaggctgc tgcttcgcca cttgctgctt caccacgaag 241 gagttcccgt gccctgggag cgggttcagg accgctgatc ggaagtgaga atcccagctg 301 tgtgtcaggg ctggaaaggg ctcgggagtg cgcggggcaa gtgaccgtgt gtgtaaagag 361 tgaggcgtat gaggctgtgt cggggcagag gcccaagatc tcannnnnnn nnnagaattt 421 ttggagtagg ctttctggct ttttaccgga aagcccctac tttctggagt ttcaaaagta 481 gactgtacgc taa SEQ ID NO: 131 U1 Promoter-Terminator_U1 snRNA Backbone LOCUS U1_promoter-term 601 bp DNA linear Accessed on 05-JUN-2025 DEFINITION synthetic linear DNA ACCESSION . Atty. Dkt. No.: 114198-6910 VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 601) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..601 / chromosome="1" / map="1p36.1" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" source 404..413 / mol_type="genomic DNA" / organism="unspecified" misc_feature 1..402 / label=U1 promoter misc_feature 403 / label=+1 A gap 404..413 / label=guide sequence / estimated_length=10 ncRNA 414..566 / gene="RNU1" / label=U1 snRNA / note="U1 small nuclear RNA; G00-119-560" / ncRNA_class="snRNA" misc_feature 567..601 / label=U1 terminator ORIGIN 1 taacacaggc taaggaccag cttctttggg agagaacaga cgcaggggcg ggagggaaaa 61 agggagaggc agacgtcact tccccttggc ggctctggca gcagattggt cggttgagtg 121 gcagaaaggc agacggggac tgggcaaggc actgtcggtg acatcacgga cagggcgact 181 tctatgtaga tgaggcagcg cagaggctgc tgcttcgcca cttgctgctt caccacgaag 241 gagttcccgt gccctgggag cgggttcagg accgctgatc ggaagtgaga atcccagctg 301 tgtgtcaggg ctggaaaggg ctcgggagtg cgcggggcaa gtgaccgtgt gtgtaaagag 361 tgaggcgtat gaggctgtgt cggggcagag gcccaagatc tcannnnnnn nnngcagggg 421 agataccatg atcacgaagg tggttttccc agggcgaggc ttatccattg cactccggat 481 gtgctgaccc ctgcgatttc cccaaatgtg ggaaactcga ctgcataatt tgtggtagtg 541 ggggactgcg ttcgcgcttt cccctgactt tctggagttt caaaagtaga ctgtacgcta 601 a Atty. Dkt. No.: 114198-6910 SEQ ID NO: 132 Lentiviral Vector Containing Ef1a Core eGFO-PuroR Cassette LOCUS Lentiviral_empty 9840 bp DNA circular SYN 17-MAR-202 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9840) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com COMMENT LentiCRISPR v2 was modified into an all-in-one dox inducible system. The addition of doxycycline induces Cas9-2A-eGFP. The U6 promoter drives constitutive sgRNA expression.. [1] Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer. Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 10.3390 / cancers11020251. Barger CJ, Branick C, Chee L, Karpf AR (pmid:30795624) DIgest TLCV2 DIg with XhoI and NheI 2x PCR, one for EF1 alph + puro and one for EGF FEATURES Location / Qualifiers source 1..9840 / mol_type="other DNA" / organism="synthetic DNA construct" CDS complement(171..962) / label=AmpR / note="label: AmpR note: note: confers resistance to ampicillin, carbenicillin, and related antibiotics transl_table: 1 codon_start: 1 translation: MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGYIELDLNSGKILESF RPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHL TDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPEL NEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSAL PAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW* gene: bla product: beta-lactamase" CDS complement(963..1031) / label=AmpR / note="label: AmpR note: note: confers resistance to ampicillin, carbenicillin, and related antibiotics transl_table: 1 codon_start: 1 translation: MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGYIELDLNSGKILESF Atty. Dkt. No.: 114198-6910 RPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGM TVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIP NDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGW FIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW * gene: bla product: beta-lactamase" promoter complement(1032..1136) / label=AmpR promoter / note="label: AmpR promoter note: gene: bla" enhancer 1402..1781 / label=CMV enhancer / note="label: CMV enhancer note: note: human cytomegalovirus immediate early enhancer" promoter 1783..1981 / label=CMV promoter / note="label: CMV promoter note: note: human cytomegalovirus (CMV) immediate early promoter" LTR 1999..2179 / label=5' LTR (truncated) / note="label: 5' LTR (truncated) note: note: truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 2226..2351 / label=HIV-1 Psi / note="label: HIV-1 Psi note: note: packaging signal of human immunodeficiency virus type 1" misc_feature 2844..3077 / label=RRE / note="label: RRE note: note: The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 3262..3306 / label=gp41 peptide / note="label: gp41 peptide note: note: recognized by the 2H10 single-chain llama nanobody transl_table: 1 codon_start: 1 translation: KNEQELLELDKWASL product: antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" misc_feature 3604..3721 / label=cPPT / CTS / note="label: cPPT / CTS note: note: central polypurine tract and central termination sequence of HIV-1" misc_feature 3766..3790 / label=Gibson / note="label: Gibson" primer_bind 3773..3789 / label=M13 fwd Atty. Dkt. No.: 114198-6910 / note="label: M13 fwd note: note: common sequencing primer, one of multiple similar variants" misc_feature 3791..4389 / label=Cutout for RNA vector insertion misc_feature 4390..4414 / label=Gibson / note="label: Gibson" primer_bind complement(4437..4453) / label=M13 rev / note="label: M13 rev note: note: common sequencing primer, one of multiple similar variants" misc_feature 4453 / label=G-->A / note="label: G-->A" promoter 4463..4674 / label=EF-1-alpha core promoter / note="label: EF-1-alpha core promoter note: note: core promoter for human elongation factor EF-1-alpha" misc_feature 4472 / label=G-->A / note="label: G-->A" CDS 4696..5292 / label=PuroR / note="label: PuroR note: note: confers resistance to puromycin transl_table: 1 codon_start: 1 translation: MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTR VGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEG LLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRN LPFYERLGFTVTADVEVPEGPRTWCMTRKPGA gene: pac from Streptomyces alboniger product: puromycin N-acetyltransferase" CDS 5302..5358 / label=P2A / note="label: P2A note: note: Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides. transl_table: 1 codon_start: 1 translation: ATNFSLLKQAGDVEENPGP product: 2A peptide from porcine teschovirus-1 polyprotein" CDS 5359..5361 / label=EGFP / note="label: EGFP note: note: mammalian codon-optimized transl_table: 1 codon_start: 1 translation: V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDE Atty. Dkt. No.: 114198-6910 LYK* product: the original enhanced GFP (Yang et al., 1996)" CDS 5359..5361 / label=EGFP / note="label: EGFP note: transl_table: 1 codon_start: 1 translation: V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDE LYK product: enhanced GFP" CDS 5362..6075 / label=EGFP / note="label: EGFP note: note: mammalian codon-optimized transl_table: 1 codon_start: 1 translation: V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDE LYK* product: the original enhanced GFP (Yang et al., 1996)" CDS 5362..6072 / label=EGFP / note="label: EGFP note: transl_table: 1 codon_start: 1 translation: V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDE LYK product: enhanced GFP" misc_feature 6096..6684 / label=WPRE / note="label: WPRE note: note: woodchuck hepatitis virus posttranscriptional regulatory element" CDS complement(6567..6578) / label=Factor Xa site / note="label: Factor Xa site note: transl_table: 1 codon_start: 1 translation: IEGR product: Factor Xa recognition and cleavage site" LTR 6756..6989 / label=3' LTR (Delta-U3) / note="label: 3' LTR (Delta-U3) note: note: self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 7021..7245 Atty. Dkt. No.: 114198-6910 / label=bGH poly(A) signal / note="label: bGH poly(A) signal note: note: bovine growth hormone polyadenylation signal" rep_origin 7291..7719 / label=f1 ori / note="label: f1 ori note: note: f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis direction: RIGHT" promoter 7733..8062 / label=SV40 promoter / note="label: SV40 promoter note: note: SV40 enhancer and early promoter" rep_origin 7913..8048 / label=SV40 ori / note="label: SV40 ori note: note: SV40 origin of replication" promoter 8110..8157 / label=EM7 promoter / note="label: EM7 promoter note: note: synthetic bacterial promoter " CDS 8176..8550 / label=BleoR / note="label: BleoR note: note: confers resistance to bleomycin, phleomycin, and Zeocin(TM) transl_table: 1 codon_start: 1 translation: MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDVTLFIS AVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGREF ALRDPAGNCVHF VAEEQD* gene: Sh ble from Streptoalloteichus hindustanus product: antibiotic-binding protein" polyA_signal 8680..8801 / label=SV40 poly(A) signal / note="label: SV40 poly(A) signal note: note: SV40 polyadenylation signal" primer_bind complement(8850..8866) / label=M13 rev / note="label: M13 rev note: note: common sequencing primer, one of multiple similar variants" protein_bind 8874..8890 / label=lac operator / note="label: lac operator note: note: The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG). bound_moiety: lac repressor encoded by lacI" promoter complement(8898..8904) / label=lac promoter Atty. Dkt. No.: 114198-6910 / note="label: lac promoter note: note: promoter for the E. coli lac operon" promoter complement(8905..8922) / label=lac promoter / note="label: lac promoter note: note: promoter for the E. coli lac operon" promoter complement(8923..8928) / label=lac promoter / note="label: lac promoter note: note: promoter for the E. coli lac operon" protein_bind 8943..8964 / label=CAP binding site / note="label: CAP binding site note: note: CAP binding activates transcription in the presence of cAMP. bound_moiety: E. coli catabolite activator protein" complement(9252..9840) / direction=LEFT / label=ori / note="direction: LEFT label: ori note: note: high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication direction: LEFT" 1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt 1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg 1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta 1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg Atty. Dkt. No.: 114198-6910 1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1981 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 2041 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 2101 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 2161 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag 2221 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc 2281 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt 2341 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa 2401 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag 2461 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg 2521 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag 2581 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag 2641 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc 2701 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa 2761 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag 2821 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga 2881 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt 2941 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa 3001 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta 3061 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct 3121 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc 3181 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa 3241 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca 3301 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg 3361 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga 3421 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc 3481 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga 3541 ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca 3601 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 3661 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 3721 tcgggtttat tacagggaca gcagagatcc agtttggtta attaacgacg ttgtaaaacg 3781 acggccagtg ccctttatgc gattcaaggt gcatatggaa ggatcagtca atggccacga 3841 atttgagatc gaaggtgtgg gggaaggaaa accctacgaa gggactcaga ctgctaagct 3901 gcaggtgacg aaaggtggcc ctcttccttt cgcgtgggac attctttccc cacagttctt 3961 ttatgggtct aaggcctata tcaaacaccc cgccgacata cccgattacc tcaagcaaag 4021 ttttcctgaa ggctttaagt gggagagagt catgaatttc gaggatgggg gcgtcgtgac 4081 cgtgacccag gactccagcc tgcaggatgg caccttaata tatcatgtta aattcatcgg 4141 agttaacttc ccaagcgatg gtccggtgat gcaaaagaaa acactaggtt gggagccatc 4201 tacagagcgc aactacccac gtgacggcgt gctcaagggg gagaaccata tggcattgaa 4261 actgaaggga ggagggcact acctctgcga gttcaaatca atctatatgg ctaaaaagcc Atty. Dkt. No.: 114198-6910 4321 ggttaagctg cctggctatc attacgtaga ctacaaactg gatattacca gccacaatga 4381 agattacacg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca 4441 tagctgtttc ctcctagcat ccgggcagag cacacatcgc ccacagtccc cgagaagttg 4501 gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa 4561 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt 4621 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acagactgcg 4681 atcgcaatgt acagtatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 4741 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 4801 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 4861 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 4921 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 4981 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg 5041 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag 5101 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg 5161 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 5221 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 5281 aagcccggtg ccggttccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc 5341 gaagagaatc ctggaccggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 5401 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 5461 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 5521 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 5581 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 5641 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 5701 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 5761 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 5821 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 5881 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 5941 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 6001 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 6061 gagctgtaca agtaactcga gcgcgttaag tcgacaatca acctctggat tacaaaattt 6121 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 6181 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 6241 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 6301 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 6361 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 6421 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 6481 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc 6541 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 6601 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 6661 tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt 6721 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg 6781 aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg 6841 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 6901 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 6961 cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga tcagcctcga 7021 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 7081 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc Atty. Dkt. No.: 114198-6910 7141 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 7201 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 7261 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 7321 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 7381 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 7441 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 7501 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 7561 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 7621 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 7681 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 7741 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 7801 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 7861 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 7921 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 7981 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 8041 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 8101 tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag 8161 gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 8221 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 8281 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 8341 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 8401 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 8461 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 8521 gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc 8581 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 8641 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 8701 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 8761 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 8821 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 8881 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 8941 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 9001 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 9061 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 9121 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 9181 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 9241 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 9301 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 9361 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 9421 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 9481 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 9541 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 9601 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 9661 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 9721 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 9781 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa SEQ ID NO: 133 Atty. Dkt. No.: 114198-6910 CFTR PTC Dual Luciferase Reporter LOCUS CFTR_PTC_dual_lu3859 bp DNA linear Accessed on 03-JUN-2025 DEFINITION natural linear DNA. ACCESSION . VERSION . KEYWORDS . SOURCE natural DNA sequence ORGANISM unspecified REFERENCE 1 (bases 1 to 3859) AUTHORS MIT TITLE Direct Submission JOURNAL Exported Jun 5, 2025 from SnapGene 8.1.0 https: / / www.snapgene.com FEATURES Location / Qualifiers source 1..3859 / mol_type="genomic DNA" / organism="unspecified" source 1885..1929 / chromosome="7" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" source 1930..1935 / chromosome="11" / mol_type="genomic DNA" / db_xref="taxon:9606" / organism="Homo sapiens" enhancer 1..380 / label=CMV enhancer / note="human cytomegalovirus immediate early enhancer" promoter 381..584 / label=CMV promoter / note="human cytomegalovirus (CMV) immediate early promoter" misc_feature 696..720 / label=Gibson misc_feature 721..726 / label=Kozak CDS 727..1836 / codon_start=1 / label=hRLuc / translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV KGLHFSQEDAPDEMGKYIKSFVERVLKNEQNSACKNWFSSLSHFVIHLNSHGFPPEVEE QAAGTLPMSCAQESGMDRHPAACASARINV" CDS 1660..1836 / codon_start=1 / label=degron Atty. Dkt. No.: 114198-6910 / translation="NSACKNWFSSLSHFVIHLNSHGFPPEVEEQAAGTLPMSCAQESGM DRHPAACASARINV" CDS 1837..1884 / codon_start=1 / label=XTEN linker / translation="SGSETPGTSESATPES" mRNA 1885..1929 / gene="CFTR" / gene_synonym="ABC35; ABCC7; CF; CFTR / MRP; dJ760C5.1; MRP7; TNR-CFTR" / product="CF transmembrane conductance regulator" / label=CFTR mRNA / note="Derived by automated computational analysis using gene prediction method: BestRefSeq." CDS 1885..1929 / codon_start=1 / gene="CFTR" / gene_synonym="ABC35; ABCC7; CF; CFTR / MRP; dJ760C5.1; MRP7; TNR-CFTR" / product="cystic fibrosis transmembrane conductance regulator" / label=CFTR / note="Derived by automated computational analysis using gene prediction method: BestRefSeq." misc_feature 1885..1929 / label=reporter_site misc_feature 1906..1908 / label=W1282X CDS 1930..1935 / codon_start=1 / label=GS / translation="GS" CDS 1936..3585 / codon_start=1 / label=hFLuc / translation="ADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM Atty. Dkt. No.: 114198-6910 YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF VDEVPKGLTGKLDARKIREILIKAKKGGKIAV" polyA_signal 3635..3859 / label=bGH poly(A) signal / note="bovine growth hormone polyadenylation signal" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacgggccc tctagactcg agcggccgcc actgtgctgg atatctgcag 721 gccaccatgg cttccaaggt gtacgacccc gagcaacgca aacgcatgat cactgggcct 781 cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact ccttcatcaa ctactatgat 841 tccgagaagc acgccgagaa cgccgtgatt tttctgcatg gtaacgctgc ctccagctac 901 ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta gatgcatcat ccctgatctg 961 atcggaatgg gtaagtccgg caagagcggg aatggctcat atcgcctcct ggatcactac 1021 aagtacctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat ctttgtgggc 1081 cacgactggg gggcttgtct ggcctttcac tactcctacg agcaccaaga caagatcaag 1141 gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt cctgggacga gtggcctgac 1201 atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt gcttgagaat 1261 aacttcttcg tcgagaccat gctcccaagc aagatcatgc ggaaactgga gcctgaggag 1321 ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc taccctctcc 1381 tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca gattgtccgc 1441 aactacaacg cctaccttcg ggccagcgac gatctgccta agatgttcat cgagtccgac 1501 cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa caccgagttc 1561 gtgaaggtga agggcctcca cttcagccag gaggacgctc cagatgaaat gggtaagtac 1621 atcaagagct tcgtggagcg cgtgctgaag aacgagcaga attctgcttg caagaactgg 1681 ttcagtagct taagccactt tgtgatccac cttaacagcc acggcttccc tcccgaggtg 1741 gaggagcagg ccgccggcac cctgcccatg agctgcgccc aggagagcgg catggataga 1801 caccctgctg cttgcgccag cgccaggatc aacgtcagcg gaagtgagac accgggtacg 1861 agtgagtcag ctactccaga aagtgattca ataactttgc aacagtgaag gaaagccttt 1921 ggagtgatag gttctgccga tgctaagaac attaagaagg gccctgctcc cttctaccct 1981 ctggaggatg gcaccgctgg cgagcagctg cacaaggcca tgaagaggta tgccctggtg 2041 cctggcacca ttgccttcac cgatgcccac attgaggtgg acatcaccta tgccgagtac 2101 ttcgagatgt ctgtgcgcct ggccgaggcc atgaagaggt acggcctgaa caccaaccac 2161 cgcatcgtgg tgtgctctga gaactctctg cagttcttca tgccagtgct gggcgccctg 2221 ttcatcggag tggccgtggc ccctgctaac gacatttaca acgagcgcga gctgctgaac 2281 agcatgggca tttctcagcc taccgtggtg ttcgtgtcta agaagggcct gcagaagatc 2341 ctgaacgtgc agaagaagct gcctatcatc cagaagatca tcatcatgga ctctaagacc Atty. Dkt. No.: 114198-6910 2401 gactaccagg gcttccagag catgtacaca ttcgtgacat ctcatctgcc tcctggcttc 2461 aacgagtacg acttcgtgcc agagtctttc gacagggaca aaaccattgc cctgatcatg 2521 aacagctctg ggtctaccgg cctgcctaag ggcgtggccc tgcctcatcg caccgcctgt 2581 gtgcgcttct ctcacgcccg cgaccctatt ttcggcaacc agatcatccc cgacaccgct 2641 attctgagcg tggtgccatt ccaccacggc ttcggcatgt tcaccaccct gggctacctg 2701 atttgcggct ttcgggtggt gctgatgtac cgcttcgagg aggagctgtt cctgcgcagc 2761 ctgcaagact acaaaattca gtctgccctg ctggtgccaa ccctgttcag cttcttcgct 2821 aagagcaccc tgatcgacaa gtacgacctg tctaacctgc acgagattgc ctctggcggc 2881 gccccactgt ctaaggaggt gggcgaagcc gtggccaagc gctttcatct gccaggcatc 2941 cgccagggct acggcctgac cgagacaacc agcgccattc tgattacccc agagggcgac 3001 gacaagcctg gcgccgtggg caaggtggtg ccattcttcg aggccaaggt ggtggacctg 3061 gacaccggca agaccctggg agtgaaccag cgcggcgagc tgtgtgtgcg cggccctatg 3121 attatgtccg gctacgtgaa taaccctgag gccacaaacg ccctgatcga caaggacggc 3181 tggctgcact ctggcgacat tgcctactgg gacgaggacg agcacttctt catcgtggac 3241 cgcctgaagt ctctgatcaa gtacaagggc taccaggtgg ccccagccga gctggagtct 3301 atcctgctgc agcaccctaa cattttcgac gccggagtgg ccggcctgcc cgacgacgat 3361 gccggcgagc tgcctgccgc cgtcgtcgtg ctggaacacg gcaagaccat gaccgagaag 3421 gagatcgtgg actatgtggc cagccaggtg acaaccgcca agaagctgcg cggcggagtg 3481 gtgttcgtgg acgaggtgcc caagggcctg accggcaagc tggacgcccg caagatccgc 3541 gagatcctga tcaaggctaa gaaaggcggc aagatcgccg tgtaagatcc gagctcggta 3601 ccaagcttaa gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 3661 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 3721 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 3781 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 3841 gatgcggtgg gctctatgg / / Clauses Clause 1. A targeted pseudouridylation polynucleotide comprising: a. an engineered H / ACA box snoRNA linked to one or two or more guide RNA targeting a eukaryotic RNA transcript, b. one or more U snRNA polynucleotides; and c. one or more RNA linker sequences linking the engineered H / ACA box snoRNA to the U snRNA polynucleotides. Clause 2. The targeted pseudouridylation polynucleotide of clause 1, wherein the targeted eukaryotic RNA transcript comprises a pre-mRNA, an mRNA, a long noncoding RNA, an enhancer RNA, or any other RNA found in the nucleoplasm of cells that is not a ribosomal RNA. Clause 3. The targeted pseudouridylation polynucleotide of clause 1 or 2, wherein the one or more U snRNA polynucleotides are from about 20 nucleotides to about100 nucleotides in length. Clause 4. The targeted polynucleotide of clause 3, wherein the one or more U snRNA polynucleotides are selected from a U1 snRNA, a U2 snRNA, a U4 snRNA, a U5 snRNA, a U6 snRNA, a U7 snRNA, a U7smOPT snRNA, U11 a snRNA, a U12 snRNA, or an Atty. Dkt. No.: 114198-6910 equivalent thereof having at least 70 sequence identity across the full length of the polynucleotide to a polynucleotide. Clause 5. The targeted polynucleotide of any one of clauses 1-3, wherein the one or more U snRNA polynucleotides comprises a U7smOPT snRNA polynucleotide of SEQ ID NO: 5, or an equivalent thereof having at least 70 sequence identity across the full length of the polynucleotide to SEQ ID NO: 5. Clause 6. The targeted pseudouridylation polynucleotide of any one of clauses 1-5, wherein the H / ACA box polynucleotide is selected from a polynucleotide shown in rows 14- 18 of Table 1. Clause 7. The targeted polynucleotide of any one of clauses 1-6, wherein the guide RNA is not linked to the 5' or 3' end of the H / ACA box sno RNA polynucleotide. Clause 8. The targeted pseudouridylation polynucleotide of any of clauses 1-7, wherein the U snRNA components linked to the 5′ end of the engineered H / ACA snoRNA polynucleotide, the 3′ end of the engineered H / ACA snoRNA polynucleotide, or both, or a mixture of both. Clause 9. The targeted pseudouridylation polynucleotide of any one of clauses 1-8, wherein the engineered H / ACA box snoRNA linked to a guide RNA targeting the eukaryotic RNA transcript further comprises a promoter polynucleotide and / or a termination polynucleotide, optionally wherein the promoter is a U1 promoter or a U7 promoter. Clause 10. The targeted pseudouridylation polynucleotide of any one of clauses 1-9, wherein the guide RNA targets an RNA transcript encoding a cystic fibrosis transmembrane receptor polypeptide (CFTR) or a myosin polypeptide. Clause 11. The targeted polynucleotide of clause 9, wherein the engineered H / ACA box snoRNA and the one or more U snRNA polynucleotides comprise a sequence shown in any one of rows 1-6 of Table 1. Clause 12. The targeted pseudouridylation polynucleotide of any one of clauses 1-11, wherein the one or more RNA linkers comprise from about 1 nucleotide to about 20 nucleotides. Atty. Dkt. No.: 114198-6910 Clause 13. The targeted pseudouridylation polynucleotide of clause 12, wherein the linker comprise at least 70% nucleotides selected from guanines, cytosines, or a mixture of both, across the full length of the linker. Clause 14. The targeted pseudouridylation polynucleotide of any one of clauses 1-13, wherein the guide RNA is selected from a polynucleotide shown in any one of rows 1-13 of Table 2. Clause 15. The targeted pseudouridylation polynucleotide of any one of clauses 1-14, wherein the polynucleotide comprises or consist of DNA or RNA. Clause 16. An isolated DNA polynucleotide encoding the targeted pseudouridylation of any one of clauses 1-15, wherein the polynucleotide comprises deoxyribonucleic acid (DNA). Clause 17. A vector or an isolated host cell comprising the isolated polynucleotide of any one of clauses 1-16. Clause 18. The vector of clause 17, wherein the vector is selected from a plasmid, a viral vector, an adenoviral associated vector (AAV), a micelle, a lipid or a lipid nanoparticle (LNP). Clause 19. The vector of clause 18, wherein the vector is a lipid nanoparticle. Clause 20. The host cell of clause 19, wherein the isolated host cell is a prokaryotic or eukaryotic cell. Clause 21. The isolated host cell of clause 20, wherein the host cell is a eukaryotic cell. Clause 22. A method to deliver a targeted pseudouridylation polynucleotide to a cell comprising contacting the cell with the isolated targeted pseudouridylation polynucleotide of any one or more of clauses 1-16, or the vector of any one of claims 17-19. Clause 23. The method of clause 22, wherein the contacting is in vitro or in vivo. Clause 24. The method of clause 22 or 23, wherein the cell is a eukaryotic cell. Clause 25. The method of clause 24, wherein the eukaryotic cell, optionally an animal or a human cell. Atty. Dkt. No.: 114198-6910 Clause 26. A method to deliver a targeted pseudouridylation polynucleotide to an animal comprising administering to the animal the isolated targeted pseudouridylation molecule of any one of clauses 1-16, or the vector of any one of claims 17-19. Clause 27. The method of clause 26, wherein the animal is a human. Equivalents Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, Appendices, patents, and other references mentioned herein or attached hereto are expressly incorporated by reference in their entirety, to the same Atty. Dkt. No.: 114198-6910 extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. Other embodiments are set forth within the following claims.

[0003] Atty. Dkt. No.: 114198-6910 REFERENCES 1. Song, J., Zhuang, Y. & Yi, C. Programmable RNA base editing via targeted modifications. Nat Chem Biol 20, 277-290 (2024). 2. Mort, M., Ivanov, D., Cooper, D.N. & Chuzhanova, N.A. A meta-analysis of nonsense mutations causing human genetic disease. Hum Mutat 29, 1037-1047 (2008). 3. Havens, M.A. & Hastings, M.L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res 44, 6549-6563 (2016). 4. Howard, M., Frizzell, R.A. & Bedwell, D.M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat Med 2, 467-469 (1996). 5. Welch, E.M. et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 447, 87-91 (2007). 6. Albers, S. et al. Engineered tRNAs suppress nonsense mutations in cells and in vivo. Nature 618, 842-848 (2023). 7. Porter, J.J., Heil, C.S. & Lueck, J.D. Therapeutic promise of engineered nonsense suppressor tRNAs. Wiley Interdiscip Rev RNA 12, e1641 (2021). 8. Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol Cell Biol 17, 83-96 (2016). 9. Reautschnig, P. et al. CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo. Nat Biotechnol 40, 759-768 (2022). 10. Katrekar, D. et al. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs. Nat Biotechnol 40, 938-945 (2022). 11. Yi, Z. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat Biotechnol 40, 946-955 (2022). 12. Eggington, J.M., Greene, T. & Bass, B.L. Predicting sites of ADAR editing in double- stranded RNA. Nat Commun 2, 319 (2011). Atty. Dkt. No.: 114198-6910 Borchardt, E.K., Martinez, N.M. & Gilbert, W.V. Regulation and Function of RNA Pseudouridylation in Human Cells. Annu Rev Genet 54, 309-336 (2020). Kufel, J. & Grzechnik, P. Small Nucleolar RNAs Tell a Different Tale. Trends Genet 35, 104-117 (2019). Karijolich, J. & Yu, Y.T. Converting nonsense codons into sense codons by targeted pseudouridylation. Nature 474, 395-398 (2011). Song, J. et al. CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons. Mol Cell 83, 139-155 e139 (2023). Adachi, H. et al. Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes. Mol Cell 83, 637-651 e639 (2023). Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013). Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823- 826 (2013). Gadgil, A. & Raczynska, K.D. U7 snRNA: A tool for gene therapy. J Gene Med 23, e3321 (2021). Rogalska, M.E. et al. Therapeutic activity of modified U1 core spliceosomal particles. Nat Commun 7, 11168 (2016). Raitskin, O., Cho, D.S., Sperling, J., Nishikura, K. & Sperling, R. RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery. Proc Natl Acad Sci U S A 98, 6571-6576 (2001). Ietswaart, R. et al. Genome-wide quantification of RNA flow across subcellular compartments reveals determinants of the mammalian transcript life cycle. Mol Cell 84, 2765-2784 e2716 (2024). Flanigan, K.M. et al. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene. Hum Mutat 32, 299-308 (2011). Atty. Dkt. No.: 114198-6910 Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014). Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 10, 1523 (2019). Vaquero-Garcia, J. et al. RNA splicing analysis using heterogeneous and large RNA- seq datasets. Nat Commun 14, 1230 (2023). Patel, S.B. & Bellini, M. The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res 36, 6482-6493 (2008). Ngo, L.H. et al. Nuclear export of circular RNA. Nature 627, 212-220 (2024). Hsiao, Y.E. et al. RNA editing in nascent RNA affects pre-mRNA splicing. Genome Res 28, 812-823 (2018). Winter, J. et al. Targeted exon skipping with AAV-mediated split adenine base editors. Cell Discov 5, 41 (2019). Dai, Q. et al. Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nat Biotechnol 41, 344-354 (2023). Pederiva, C. et al. Control of protein synthesis through mRNA pseudouridylation by dyskerin. Sci Adv 9, eadg1805 (2023). Matera, A.G. & Wang, Z. A day in the life of the spliceosome. Nat Rev Mol Cell Biol 15, 108-121 (2014). Cao, Y. et al. RNA-based translation activators for targeted gene upregulation. Nat Commun 14, 6827 (2023). Sun, Y. et al. Improved RNA base editing with guide RNAs mimicking highly edited endogenous ADAR substrates. Nat Biotechnol (2025). Choi, J. et al.2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation. Nat Struct Mol Biol 25, 208-216 (2018). Elliott, B.A. et al. Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo. Nat Commun 10, 3401 (2019). Atty. Dkt. No.: 114198-6910 Arango, D. et al. Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Mol Cell 82, 2797-2814 e2711 (2022). Thalalla Gamage, S. et al. Antisense pairing and SNORD13 structure guide RNA cytidine acetylation. RNA 28, 1582-1596 (2022).

Claims

Atty. Dkt. No.: 114198-6910 WHAT IS CLAIMED IS:

1. A targeted pseudouridylation polynucleotide comprising: a. an engineered H / ACA box snoRNA linked to one or more, or two or more guide RNA targeting a eukaryotic RNA transcript, b. one or more U snRNA polynucleotides; and c. one or more RNA linker sequences linking the engineered H / ACA box snoRNA to the U snRNA polynucleotides.

2. The targeted pseudouridylation polynucleotide of claim 1, wherein the targeted eukaryotic RNA transcript comprises a pre-mRNA, an mRNA, a long noncoding RNA, an enhancer RNA, or any other RNA found in the nucleoplasm of cells that is not a ribosomal RNA.

3. The targeted pseudouridylation polynucleotide of claim 1 or 2, wherein the one or more U snRNA polynucleotides are from about 20 nucleotides to about100 nucleotides in length.

4. The targeted polynucleotide of claim 3, wherein the one or more U snRNA polynucleotides are selected from a U1 snRNA, a U2 snRNA, a U4 snRNA, a U5 snRNA, a U6 snRNA, a U7 snRNA, a U7smOPT snRNA, U11 a snRNA, a U12 snRNA, or an equivalent thereof having at least 70 sequence identity across the full length of the polynucleotide to a polynucleotide.

5. The targeted polynucleotide of any one of claims 1-3, wherein the one or more U snRNA polynucleotides comprises a U7smOPT snRNA polynucleotide of SEQ ID NO: 5, or an equivalent thereof having at least 70 sequence identity across the full length of the polynucleotide to SEQ ID NO:

5.

6. The targeted pseudouridylation polynucleotide of any one of claims 1-5, wherein the H / ACA box polynucleotide is selected from a polynucleotide shown in rows 14-18 of Table 1.Atty. Dkt. No.: 114198-6910 7. The targeted polynucleotide of any one of claims 1-6, wherein the guide RNA is not linked to the 5’ or 3’ end of the H / ACA box sno RNA polynucleotide.

8. The targeted pseudouridylation polynucleotide of any of claims 1-7, wherein the U snRNA components linked to the 5′ end of the engineered H / ACA snoRNA polynucleotide, the 3′ end of the engineered H / ACA snoRNA polynucleotide, or both, or a mixture of both.

9. The targeted pseudouridylation polynucleotide of any one of claims 1-8, wherein the engineered H / ACA box snoRNA linked to a guide RNA targeting the eukaryotic RNA transcript further comprises a promoter polynucleotide and / or a termination polynucleotide, optionally wherein the promoter is a U1 promoter or a U7 promoter.

10. The targeted pseudouridylation polynucleotide of any one of claims 1-9, wherein the guide RNA targets an RNA transcript encoding a cystic fibrosis transmembrane receptor polypeptide (CFTR) or a myosin polypeptide.

11. The targeted polynucleotide of claim 9, wherein the engineered H / ACA box snoRNA and the one or more U snRNA polynucleotides comprise a sequence shown in any one of rows 1-6 of Table 1.

12. The targeted pseudouridylation polynucleotide of any one of claims 1-11, wherein the one or more RNA linkers comprise from about 1 nucleotide to about 20 nucleotides.

13. The targeted pseudouridylation polynucleotide of claim 12, wherein the linker comprise at least 70% nucleotides selected from guanines, cytosines, or a mixture of both, across the full length of the linker.

14. The targeted pseudouridylation polynucleotide of any one of claims 1-13, wherein the guide RNA is selected from a polynucleotide shown in any one of rows 1-13 of Table 2.

15. The targeted pseudouridylation polynucleotide of any one of claims 1-14, wherein the polynucleotide comprises or consist of DNA or RNA.

16. An isolated DNA polynucleotide encoding the targeted pseudouridylation of any one of claims 1-15, wherein the polynucleotide comprises deoxyribonucleic acid (DNA).Atty. Dkt. No.: 114198-6910 17. A vector or an isolated host cell comprising the isolated polynucleotide of any one of claims 1-16.

18. The vector of claim 17, wherein the vector is selected from a plasmid, a viral vector, an adenoviral associated vector (AAV), a micelle, a lipid or a lipid nanoparticle (LNP).

19. The vector of claim 18, wherein the vector is a lipid nanoparticle.

20. The host cell of claim 19, wherein the isolated host cell is a prokaryotic or eukaryotic cell.

21. The isolated host cell of claim 20, wherein the host cell is a eukaryotic cell.

22. A method to deliver a targeted pseudouridylation polynucleotide to a cell comprising contacting the cell with the isolated targeted pseudouridylation polynucleotide of any one or more of claims 1-16, or the vector of any one of claims 17-19.

23. The method of claim 22, wherein the contacting is in vitro or in vivo.

24. The method of claim 22 or 23, wherein the cell is a eukaryotic cell.

25. The method of claim 24, wherein the eukaryotic cell, optionally an animal or a human cell.

26. A method to deliver a targeted pseudouridylation polynucleotide to an animal comprising administering to the animal the isolated targeted pseudouridylation molecule of any one of claims 1-16, or the vector of any one of claims 17-19.

27. The method of claim 26, wherein the animal is a human.

Images