Targeted nuclear RNA splitting and polyadenylation with CRISPR-Cas

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application Serial No. 62 / 791,971, filed on January 14, 2019, and U.S. Provisional Application Serial No. 62 / 877,415, filed on July 23, 2019.BACKGROUND

[0002] The post-transcriptional cleavage and polyadenylation of messenger and long noncoding RNA is coordinated by a supercomplex of ~20 individual proteins within the eukaryotic nucleus (Mandel et al., Cell Mol Life Sci, 2008, 65:1099-1122; Xiang et al., Mol Cell Biol, 2014, 34:1894-1910). Polyadenylation plays an essential role in controlling RNA transcript stability, nuclear export, and translation efficiency (Colgan & Manley, Genes Dev, 1997, 11:2755-66; Guhaniyogi & Brewer, Gene, 2001, 265:11-23; Wu & Brewer, Gene, 2012, 500:10-21; Carmody &Wente, J Cell Sci, 2009, 122:1933-37). More than half of all human RNA transcripts contain multiple polyadenylation signal sequences that can undergo alternative cleavage and polyadenylation during development and cellular differentiation (Tian & Manley, Nat Rev Mol Cell Biol, 2017, 18:18-30; Elkon et al., Nat Rev Genet, 2013, 14:496-506). Alternative cleavage and polyadenylation is an important mechanism for the control of gene expression and defects in 3' end processing can give rise to myriad human diseases (Chang et al., Endocrinol Metab, 2017, 32:413-21; Curinha et al., Nucleus, 2014, 5:508-19).

[0003] Zahir Ali et al. "CRISPR / Cas13 as a Tool for RNA Interference", Trends in Plant Science, vol. 23, no. 5, 28 March 2018 discusses the use of CRISPR / Cas13 in plants for various types of RNA manipulation.

[0004] Thus, there is a need in the art for compositions and methods for modulating alternative cleavage and polyadenylation. The present invention satisfies this need.SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a fusion protein comprising (a) a CRISPR-associated (Cas) protein; and (b) a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21. In one embodiment, the Cas protein is catalytically dead Cas13 (dCas13). In one embodiment, dCas13 comprises SEQ ID NO:47 or a variant thereof. In one embodiment, NUDT21 comprises SEQ ID NO:51 or a variant thereof.

[0006] In one embodiment, the fusion protein further comprises a nuclear localization signal (NLS). In one embodiment, the NLS comprises SEQ ID NO:75 or a variant thereof. In one embodiment, the fusion protein comprises SEQ ID NO:698 or a variant thereof.

[0007] In one embodiment, the invention provides a method of modulating the cleavage, polyadenylation or both of an RNA transcript in vitro. In one embodiment, the invention provides a fusion protein of the invention or the nucleic acid molecule encoding a method of the invention and a guide nucleic acid comprising a sequence complimentary to a target RNA sequence in the RNA transcript for use in in vivo modulation of the cleavage, polyadenylation or both of an RNA transcript in a subject.

[0008] In one embodiment, the invention provides a nucleic acid encoding a fusion protein of the invention.

[0009] In one embodiment, the invention provides the fusion protein of the invention or a nucleic acid molecule of the invention for use as a medicament. In one embodiment, the invention provides the fusion protein of the invention or a nucleic acid molecule of the invention for use in the treatment of human RNA processing diseases.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1, comprising Figure 1A and Figure 1B depicts the design and expression of fusion proteins encoding catalytically dead Cas13 and human polyadenylation complex factors. Figure 1A depicts a diagram of the vectors encoding fusions between catalytically dead PspCas13b and components of the mammalian polyadenylation supercomplex, CPSF30, WDR33, and NUDT21. F - 3xFLAG epitope; NLS - Ty1 nuclear localization sequence; pA - SV40 polyadenylation sequence. Figure 1B depicts immunohistochemistry using a primary anti-FLAG antibody and an Alexa488 conjugated secondary antibody detecting the nuclear localization of the dPspCas13b fusion proteins expressed in mammalian COS7 cells. Scale bars in B, 10 µm. Figure 2, comprising Figure 2A through Figure 2H, depicts the design and generation of a fluorescent reporter for RNA cleavage and polyadenylation in mammalian cells. Figure 2A depicts a diagram of the sfGFPapa reporter plasmid. Figure 2B depicts the green fluorescent protein superfolder GFP (sfGFP) which forms a beta barrel composed of 11 beta strands. Removal of the 11 th< beta strand abolishes fluorescence. Figure 2C depicts transcription of the sfGFPapa reporter and processing of the resulting transcript which removes the coding sequence of the 11 th< sfGFP beta strand. Figure 2D demonstrates that the removal of the 11 th< beta strand results in a lack of fluorescent signal in mammalian cells. Figure 2E depicts a diagram of the sfGFPapa-pA reporter plasmid, which contains an upstream polyadenylation sequence. Figure 2F is a schematic depicting that sfGFP can tolerate linker sequences between the 10 th< and 11 th< beta strands without abolishing fluorescence. Figure 2G depicts transcription of the sfGFPapa-pA reporter and processing of the resulting transcript. Figure 2H demonstrates that processed transcripts result in robust green fluorescence due to translation of the sfGFP(1-10)-L-(11) functional open reading frame. Scale bars in Figure 2D and Figure 2H, 100 µm. Figure 3, comprising Figure 3A through Figure 3F, depicts CRISPR-Cas13-mediated cleavage and polyadenylation of a reporter mRNA in mammalian cells. Figure 3A depicts a diagram showing the Postscriptr targeting strategy to induce alternative cleavage and polyadenylation of the sfGFPapa reporter construct. A crRNA was designed to target an intronic sequence downstream of the coding sequence of the 11 th< beta strand of sfGFP. Figure 3B depicts expression of the dPspCas13b-NUDT21 fusion protein and demonstrates that intronic targeting crRNA resulted in green fluorescent cells after 24 hours. Figure 3C depicts 3'RACE products amplified from cells expressing dPspCas13b-NUDT21 fusion protein and non-targeting control or intronic targeting crRNA. Figure 3D and Figure 3E depict sequences of the five most proximal 3'RACE products relative to the crRNA target site. Please see Example 3 for complete list of 3'RACE sequences. Green underlined and lowercase nucleotides highlight 3' non-templated nucleotide addition. Figure 3F depicts nucleotide frequencies at Postscriptr-mediated cleavage and polyadenylation sites. Figure 4, comprising Figure 4A through Figure 4I, depicts Postscriptr-mediated alternative cleavage and polyadenylation of human SREBP1 in HEK293T cells. Figure 4A depicts a diagram of the SREBP1 maturation pathway. Figure 4B depicts the human SREBP1 locus contains an intronic PAS between exon 7 and exon 8, which results in translation of an SREBP1Δ isoform which terminates translation adjacent to the normal S2P cleavage site. Figure 4C depicts the SREP1 sequence and SREBP1Δ isoform sequence. Figure 4D depicts a diagram of the Postscriptr-mediated targeting strategy and sequence of the recovered Postscriptr-induced cleaved transcript. Figure 4E depicts a diagram of the Postscriptr-mediated targeting strategy and sequence of the recovered Postscriptr-induced polyadenylated transcript. Figure 4F depicts the predicted translational stop. Figure 4G depicts quantitative realtime-PCR (qRT-PCR) gene expression analysis of SREBP1 transcript levels upstream of the crRNA target sequence in HEK293T cells targeted with a non-targeted or SREBP1-targeted crRNA. Figure 4H depicts quantitative realtime-PCR (qRT-PCR) gene expression analysis of SREBP1 transcript levels spanning the crRNA target sequence in HEK293T cells targeted with a non-targeted or SREBP1-targeted crRNA. Figure 4I depicts quantitative realtime-PCR (qRT-PCR) gene expression analysis of transcript levels of the LDLR gene in HEK293T cells targeted with a non-targeted or SREBP1-targeted crRNA. Figure 5 depicts experimental results demonstrating that classic mammalian nuclear localization signals were insufficient to promote nuclear localization of dPspCas13b fusion proteins. Immunohistochemistry using a primary anti-FLAG antibody and secondary Alexa488 conjugated secondary antibody were used to detecting the localization of dPspCas13b fusion proteins expressed in mammalian COS7 cells. Fusions proteins contained either no NLS, a classic SV40 NLS or the bipartite NLS from Nucleoplasmin (NPM). Scale bars in =10 µm Figure 6, comprising Figure 6A and Figure 6B, depicts experimental results demonstrating that inhibiting splicing promotes expression of the sfGFP(1-10)-L-(11) open reading frame. Figure 6A depicts a diagram of the sfGFPapa reporter and predicted transcription and processing steps resulting from treatment with the splicing inhibitor isoginkgetin. Figure 6B depicts COS7 cells transiently transfected with the sfGFPapa reporter for 24 hours were treated with DMSO or isoginkgetin for an additional 24 hours. Cells treated with isoginkgetin resulted in detectable green fluorescence. Scale bars in B =100 µm. Figure 7, comprising Figure 7A and Figure 7B, depicts experimental results demonstrating that the dPspCas13b fusions to CPSP30 or WDR33 were not sufficient to promote cleavage and polyadenylation of the sfGFPapa reporter mRNA. Figure 7A depicts Postscriptr targeting of the sfGFPapa reporter using the dPspCas13b-CPSF30 fusion protein using an intronic-targeting crRNA did not result in detectable green fluorescence relative to a control crRNA. Figure 7B depicts Postscriptr targeting of the sfGFPapa reporter using the dPspCas13b-WDR33 fusion protein using an intronic-targeting crRNA did not result in detectable green fluorescence relative to a control crRNA. Scale bars in =100 µm Figure 8 depicts experimental results demonstrating Postscriptr-mediated alternative cleavage and polyadenylation of human SREBP1 transcripts. PCR amplified 3'RACE products from cells expressing dPspCas13b-NUDT21 with a non-targeting control or SREBP1-targeting crRNAs. Figure 9, comprising Figure 9A through Figure 9B, depicts experimental results demonstrating Cas13b crRNA Sequence Modifications Enhance Postscriptr Activity. Figure 9A depicts a schematic demonstrating CRISPR-Cas 13 guide-RNAs are typically expressed in mammalian cells from Polymerase III (Pol III) promoters, which are terminated by poly(T) sequences. Recently it has been shown that a stretch of only 4 T's can result in 75% decrease in full length expression of a small RNA by the U6 promoter. Figure 9B depicts a schematic demonstrating multiple Cas13b crRNAs contain Direct Repeat (DR) sequences. Figure 9C depicts a schematic demonstrating that the DR sequences contain a stretch of 4 or 5 T's. Figure 9D depicts crystal structures demonstrating that these nucleotides fall within the loop region, and some positions do not make direct molecular interactions. Yellow highlight in 9B and Yellow arrowhead in 9D. Figure 9E depicts a schematic demonstrating generation conservative mutations (T to C) in the DR of PspCas13b crRNA. Figure 9F depicts experimental results demonstrating the determination of their relative effectiveness in Postscriptr targeted activation of the sfGFPapa fluorescent reporter. Remarkably, two mutations T17C and T18C resulted in enhanced Postscriptr activity, whereas another mutation T19C resulted in decreased activity similar to a non-targeting (NT) guide-RNA. Figure 10 depicts a sequence alignment of NUDT21 proteins from human, fly and worm. Figure 11 depicts experimental results demonstrating the relative activities of Postscriptr fusion proteins. The impact of different sequence modifications to the dPspCas13b-NUDT21 fusion protein were assessed using activation of the sfGFPapa reporter in mammalian cells guided by an intronic targeting crRNA. Remarkably, fly and worm orthologs of NUDT21 showed comparable levels of activation relative to human, which is likely due to their high sequence conservation across species and conserved role in polyadenylation. These orthologs differ at their N-terminus, which is not only dispensable for Postscriptr-mediated activation, but resulted in enhanced activity. This may be due to the fact that acetylation of Lysines in these regions has been shown to inhibit NUDT21 activity. Mutation of residues which prevent RNA binding by NUDT21 (R63S and F103A) showed little effect on its function as a fusion to dCas13. Strikingly, a tandem dimer of NUDT21, which normally functions as an obligate dimer, resulted in markedly enhanced activation. Further, Postscriptr enzymes with a truncated C-terminus of dPspCas13 (Δ984-1090), showed similar levels of sfGFPapa activation. Figure 12 depicts a schematic showing the molecular-genetic basis for Myotonic Dystrophy Type 1 (DM1). DM1 is a monogenic autosomal dominant disorder which is characterized by progressive muscle wasting, myotonia, cardiac arrhythmias, and cognitive dysfunction. DM1 is the most common adult-onset muscular dystrophy and arises from the expansion and expression of a CUG trinucleotide repeat in the noncoding 3' untranslated region of the human Dystrophia myotonica protein kinase (DMPK) gene. Mutant DMPK mRNAs with greater than ~50 CUG repeats form toxic nuclear RNA foci, which prevent normal DMPK expression and induce widespread defects in alternative splicing and alternative polyadenylation by sequestering members of the muscleblind-like (MBNL) family of RNA binding proteins. There are no approved therapies specific for DM1 and current strategies targeting CUG RNA repeats do not address loss of DMPK expression. Figure 13, comprising Figure 13A through Figure 13D, depicts therapeutic correction of DM1 with targeted alternative cleavage and polyadenylation of human DMPK transcripts. Alternative polyadenylation (APA) of RNA is an important regulatory mechanism controlling gene expression during development and disease. Recent deep sequencing has revealed that some DMPK transcripts can be alternatively cleaved and polyadenylated at a position upstream of the site of CUG expansion, suggesting that manipulating APA could be a useful approach for both preserving DMPK expression while preventing transcription of downstream toxic repeat RNAs. Figure 13A depicts a schematic of Postscriptr. Figure 13B depicts a schematic demonstrating that Postscriptr combines the programmable RNA-targeting capability of CRISPR-Cas13 with a mammalian polyadenylation factor to induce site-specific cleavage and polyadenylation of RNA transcripts. Figure 13C depicts experimental results demonstrating Postscriptr can robustly induce alternative cleavage and polyadenylation of endogenous human DMPK transcripts upstream of the site of CUG repeat expansion. Figure 13D depicts experimental results demonstrating targeted alternative polyadenylation of mutant DMPK transcripts by Postscriptr can both rescue DMPK expression and prevent the transcription of downstream toxic CUG repeat RNA. Figure 14, comprising Figure 14A through Figure 14C, depicts the molecular origins of DM1. Figure 14A depicts a schematic demonstrating that myotonic dystrophy Type 1 (DM1) results from a microsatellite CTG repeat expansion in the 3' UTR of the human DMPK gene. Figure 14B depicts the DMPK CUG exp< RNA forms a stable hairpin, which is retained in nuclear foci, thus preventing normal DMPK gene expression and sequesters the MBNL family of RNA binding proteins. Figure 14C depicts a schematic demonstrating that the nuclear foci result in widespread defects in alternative splicing and polyadenylation. Figure 15, comprising Figure 15A through Figure 15C, depicts schematics demonstrating alternative polyadenylation. Figure 15A depicts a schematic demonstrating deep sequencing of the human transcriptome has revealed that more than half of all genes undergo alternative cleavage and polyadenylation at intronic, proximal, or distal polyadenylation signals (PAS). Figure 15B depicts a schematic demonstrating a supercomplex of ~20 proteins coordinates 3' end processing and transcriptional termination. RNA binding components of the CFIm and CPSF sub complex recognize RNA motifs in pre-RNA transcripts which specify the site of cleavage and polyadenylation. Figure 15C depicts a schematic demonstrating the novel RNA editing technology described herein, named Postscriptr, which is sufficient to induce targeted cleavage and polyadenylation using CRISPR-Cas13. Figure 16, comprising Figure 16A and Figure 16B, depicts experimental results demonstrating nuclear localization of dCas13 fusion proteins require a unique NLS. Figure 16A depicts a schematic of the vector encoding fusions between catalytically dead PspCas13b and NUDT21. Figure 16B depicts experimental results demonstrating mammalian cleavage and polyadenylation occurs in the nucleus. A non-classical nuclear localization signal (NLS) from the yeast retrotransposon Ty1 is essential for nuclear localization of the dPspCas13b-NUDT21 fusion protein. Figure 17, comprising Figure 17A and Figure 17B, depicts experimental results demonstrating targeted cleavage and polyadenylation of RNA. Figure 17A depicts a schematic showing the Postscriptr targeting strategy to induce alternative cleavage and polyadenylation of the sfGFPapa reporter construct. A crRNA was designed to target an intronic sequence downstream of the coding sequence of the 11th beta strand of sfGFP. Figure 17B depicts expression of the dPspCas13b-NUDT21 fusion protein and intronic targeting crRNA resulted in green fluorescent cells after 24 hours. Figure 18, comprising Figure 18A and Figure 18B, depicts experimental results demonstrating Postscriptr editing of endogenous human DMPK transcripts. Figure 18A depicts the design of guide-RNAs targeting the DMPK 3' UTR at a position downstream of the DMPK stop codon and upstream of the CUGexp site. Figure 18B depicts experimental results demonstrating Postscriptr editing of DMPK transcripts in HEK293T cells revealing that Postscriptr promoted the utilization of the proximal PAS and decreased expression of the distal PAS with full length DMPK protein coding sequences, indicated by retention of the distal exons. Thus, this strategy could provide a mechanism to promote DMPK expression and prevent downstream expression of CUGexp RNA. Figure 19, comprising Figure 19A through Figure 19D, depicts experimental results demonstrating targeting CUG repeat RNA foci with dCas13. Figure 19A depicts a schematic of a fusion of eGFP to dPspCas13b to visualize dCas13 subcellular localization. This construct is named HiLightr Green. Figure 19B depicts DT960, an expression plasmid which contains 960 CUG repeats in the context of human DMPK exons 11-15, which was used to induce nuclear foci. Figure 19C depicts experimental a schematic of CAGx9 guide-RNA. Figure 19D depicts experimental results demonstrating HiLightr green co-localized with mCherry-MBNL1 to RNA foci when targeted with an antisense CAGx9 guide-RNA but remained unlocalized when using a non-targeting guide-RNA. Figure 20, comprising Figure 20A and Figure 20B, depicts experimental results demonstrating CUGexp RNAs prevent mRNA expression. Figure 20A depicts luciferase expression vectors containing the human DMPK 3' UTR with either 12 CUG repeats (pGL3P-DT12a) or 960 CUG repeats (pGL3P-DT960). Figure 20B depicts experimental results demonstrating the presence of the 960 CUG repeats resulted in a 90% reduction in luciferase activity when expressed in COS7 cells. Figure 21, comprising Figure 21A through Figure 21D, depicts the inducible DM1 mouse model. An inducible humanized mouse model of DM1 is used which expresses a 960 CUG expansion RNA in the context of human DMPK exons 11-15. Figure 21A depicts a schematic demonstrating crossing a transgene to a skeletal muscle specific rtTA transgene. Figure 21B depicts a schematic demonstrating that transgene expression can be induced by doxycycline (dox). Figure 21C depicts the Postscriptr editing components encoded in a lentiviral vector. Figure 21D depicts that the lentiviral vector is used to generate lentiviral particles which are delivered during postnatal development concomitant with dox. Postscriptr editing of the CUG encoded transcripts are used to model the effectiveness of Postscriptr mitigation of DM1. Figure 22, comprising Figure 22A through Figure 22D, depicts experimental results demonstrating the development of a robust nuclear localized CRISPR-Cas13 fusion protein for the visualization of toxic RNA foci. Figure 22A depicts the design of a catalytically dead PspCas13b (dPspCas13b) encoding an N-terminal 3xFLAG and Ty1 NLS and C-terminal eGFP. F - 3xFLAG epitope; NLS - Ty1 nuclear localization sequence; pA - SV40 polyadenylation sequence. Figure 22B depicts a diagram depicting the components of the DT960 vector, which encodes a C-terminal genomic fragment of human DMPK (exons 11-15) with 960 CTG repeat expansion. Figure 22C depicts the design of the CAGx9 crRNA and its predicted targeting with CUG exp< RNA. Figure 22D depicts representative images showing the cellular localization of hilightR green targeted with either a non-targeting or CAGx9 crRNA in COS7 cells expressing CUG exp< RNA. Scale bars, 10 µm. Figure 23, comprising Figure 23A and Figure 23B, depicts experimental results demonstrating co-localization of hilightR green with CUG exp< foci and MBNL1. Figure 23A depicts immunohistochemistry using an anti-FLAG antibody was used to detect hilightR red, which co-localized with CUG exp< RNA detected using FISH, when targeted with the CAGx9 crRNA. Figure 23B depicts HilightR green co-localized with mCherry-MBNL1 in COS7 cells expressing CUG exp< RNA foci when targeted with the CAGx9 crRNA, but not with a non-targeting crRNA. Scale bars, 10 µm. Figure 24, comprising Figure 24A and Figure 24B, depicts experimental results demonstrating degradation of toxic RNA foci by CRISPR-Cas13. Figure 24A depicts coexpression of active PspCas13b encoding a Ty1 NLS (eraseR) significantly decreased the number of RNA foci in cells expressing CUG exp< RNA, when targeted with CAG crRNAs designed with target sequences in all three frames, detected by mCherry-MBNL1. Figure 24B depicts representative micrographs of cells targeted by eraseR showing foci detected by mCherry-MBNL1, which are significantly decreased in number and appear fainter. Scale bars, 10 µm. ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.0001. Figure 25, comprising Figure 25A through Figure 25C, depicts experimental results demonstrating detection of induced CUG exp< RNA foci in COS7 cells. Figure 25A depicts COS7 cells expressing 960 copies of CUG repeats induced RNA foci as detected using FISH with a CAG repeat antisense probe. AF488 - Alexa Fluor 488. Figure 25B depicts expression of CUG exp< RNA induces the localization of MBNL1 to foci, as detected using an mCherry-MBNL1 fusion protein. Figure 25C depicts localization of dPspCas13b-mCherry (hilightR red) guided by either a non-targeting or CAGx9 crRNA in COS7 cells expressing CUG exp< RNA. Scale bars, 10 µm. Figure 26 depicts experimental results demonstrating co-localization of hilightR green with splicing speckles. In agreement with previous reports, CUG exp< RNA foci marked by hilightR green targeted with a CAGx9 crRNA, co-localized with splicing speckles, as detected using an anti-SC-35 antibody. Scale bars, 10 µm. Figure 27 depicts experimental results demonstrating catalytically dead Cas13 (dCas13) does not significantly reduce the number of CUG exp< RNA foci. Expression of dPspCas13b targeted with CAGx9 crRNAs does not significantly reduce the number of CUG exp< RNA foci per cell, as detected by mCherry-MBNL1. ns - not significant. Figure 28, comprising Figure 28A through Figure 28D, depicts a diagram demonstrating therapeutic modulation of DM1 by CRISPR-Cas13. Figure 28A depicts that myotonic Dystrophy Type1 is caused by the expansion and expression of a CUG repeat in the 3' noncoding UTR of the human DMPK gene. This CUG expansion forms stable hairpin structures, which bind and sequester the MBNL family of RNA binding proteins, resulting in widespread defects in alternative splicing and polyadenylation. Figure 28B depicts CUG repeats are resistant to cleavage induced by Antisense Oligonucleotides (ASO), however, ASOs have been successfully used to block binding of MBNL1 proteins. However, many challenges remain to deliver therapeutically effective levels of ASOs to human tissues. Figure 28C depicts specific binding of dCas13 guide by a crRNA, or potentially the crRNA alone, can serve to block MBNL proteins and rescue splicing and polyadenylation defects, or when combined with a fluorescent protein, highlight CUG repeat RNA foci. Figure 28D depicts catalytically active Cas13 can be used to cleave and degrade CUG repeat RNA to prevent MBNL sequestration, as well as other potential CUG repeat-induced pathologies, such as RAN dependent translation of toxic peptides. Figure 29 depicts a schematic showing myotonic dystrophy type 1 (DM1) is an inherited multi-system, progressively debilitating disease occurring in 1 in 8,000 individuals, with an incidence as high as 1 in 500 in specific populations Cardiac complications develop in ~80% of DM1 patients and is the primary cause of death. DM1 arises from the expansion and expression of a CUG trinucleotide repeat in the noncoding 3' untranslated region of the human Dystrophia myotonica protein kinase (DMPK) gene. Mutant DMPK mRNAs with greater than ~50 CUG repeats form toxic nuclear RNA foci, which prevent normal DMPK expression and induce widespread defects in alternative splicing by sequestering members of the muscleblind-like (MBNL) family of RNA binding proteins. Due to the multitude of disrupted muscle genes underlying DM1 pathogenesis, patients often present with a variety of clinical cardiac phenotypes, including atrial and ventricular arrhythmias, dilated cardiomyopathy, and myocardial fibrosis. RNA binding CRISPR-Cas13, when localized with a robust non-classical nuclear localization signal (hilightR and eraseR), can be used to visualize and degrade toxic nuclear RNA foci in cells. Figure 30, comprising Figure 30A through Figure 30E, depicts experimental results demonstrating therapeutic rescue of heart function in a mouse model of DM1. Figure 30A depicts the generation of CUG960 cardiac DM1 mouse model. Figure 30B depicts the generation of CUG960 cardiac DM1 mouse model. Figure 30C depicts a diagram of eraseR AAV construct. Figure 30D depicts experimental results demonstrating heart-specific gene delivery and expression using AAV9. Figure 30E depicts delivery of eraseR AAV targeting CUGexp RNA reversal of the cellular and electrical abnormalities in DM1 hearts. Figure 31, comprising Figure 31A through Figure 31D, depicts experimental results demonstrating activation of Calcineurin signaling using Postscriptr. Figure 31A depicts a schematic showing calcineurin is a Ca2+ / Calmodulin activated protein phosphatase which is auto-inhibited by a C-terminal autoinhibitory domain in the absence of calcium signaling. Figure 31A depicts a schematic showing upon activation, Calcineurin dephosphorylates NFAT transcription factors, which allows for nuclear entry and activation of NFAT target genes. Figure 31A depicts a schematic showing the design of guide-RNAs to induce the alternative cleavage and polyadenylation of Calcineurin (PPP3CB) gene to allow for the expression of an N-terminal fragment of Calcineurin catalytic domain which lacks the C-terminal auto-inhibitory domain. Figure 31A depicts Postscriptr expression in mouse fibroblasts targeting the PPP3CB gene resulted in nuclear localization of an NFAT-GFP reporter gene, which is normally retained in the cytoplasm and was not affected by a non-targeting guide-RNA. Figure 32, comprising Figure 32A through Figure 32D, depicts strategies to enhance RNA visualization and fusion protein localization with dCas13. Figure 32A depicts a schematic depicting fusion of single Green Fluorescent Protein (GFP) to catalytically inactive Cas13 (dCas13), which can be used for specific visualization of nuclear RNA repeat foci in cells. Figure 32A depicts a schematic depicting fluorescent complementation inherent in fluorescent proteins (for example GFP, superfolder GFP, or superfolder Cherry) could be harnessed to reconstitute fluorescent proteins to dCas13 (for example, the complement pair sfGFP 1-10 and sfGFP11). Figure 32C depicts a schematic depicting tandem assembly of small non-fluorescent components can be used to reconstitute a large tandem array of fluorescent proteins to dCas13, which has the potential to increase the signal to noise ratio of dCas13 targeted RNAs. Figure 32A depicts a schematic depicting this approach could be similarly useful for targeting fusion proteins (Protein 'X') when co-expressed as a fusion to a complementary fluorescent fusion protein (for example, sfGFP1-10). Figure 33, comprising Figure 33A through Figure 33C, depicts the structure-function analysis of Postscriptr RNA editing. Figure 33A depicts structural modeling of the dCas13b-NUDT21 fusion protein and crRNA, using high resolution crystal structures of Cas13b (6DTD) and NUDT21 (3MDG). Figure 33B depicts results demonstrating NUDT21 forms a natural homodimer, which due to the close proximity of N and C-termini, can be expressed as a tandem dimer fused to dCas13b (dCas13b-tdNUDT21). Figure 33C depicts a model depicting the structural orientation of the dCas13b-NUDT21 fusion protein hybridized anti-sense to a Target RNA. The orientation of NUDT21 is predicted to occur 3' to the crRNA target sequence on the Target RNA, which is consistent with the observed location of Postscriptr-induced RNA cleavage and polyadenylation. DETAILED DESCRIPTION

[0011] In one aspect, the invention is based on the development of novel fusion proteins which allows for targeted RNA cleavage and polyadenylation of RNA transcripts by CRISPR-Cas. This fusion protein, termed Postscriptr herein, comprises a Cas protein, a nuclear localization signal (NLS) and a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21. Mutations in Cas13 generates a catytically dead enzyme (dCas) but retains RNA binding affinity. Thus, a fusion of dCas13 and a cleavage and / or polyadenylation protein allows for targeted cleavage and / or polyadenylation protein. Poscripter allows for non-genomic manipulation of gene expression, useful for both basic research and therapeutic applications.

[0012] Thus, in one embodiment, the invention provides compositions and methods for modulating the cleavage, polyadenylation or both of an RNA transcript in a subject. In one embodiment, the invention provides a fusion protein comprising a CRISPR-Associated (Cas) protein, and a cleavage or polyadenylation protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a linker. In one embodiment, the linker links the Cas protein and cleavage and / or polyadenylation protein. In one embodiment, the fusion protein comprises a tag.

[0013] In one embodiment, the fusion protein comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698.

[0014] In one embodiment, the invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a cleavage or polyadenylation protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a linker. In one embodiment, the linker links the Cas protein and cleavage or polyadenylation protein. In one embodiment, the fusion protein comprises a tag.

[0015] In one embodiment, nucleic acid molecule comprises a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 725-727.

[0016] Also disclosed herein are novel fusion proteins which allows for targeted nuclear RNA cleavage and degradation. This fusion protein, termed EraseR herein, comprises a Cas protein and a nuclear localization signal (NLS). CRISPR-Cas13 systems bind only to RNA and function as specific endoribonucleases to cleave target RNAs, bypassing the risk of germline editing that is associated with DNA-binding CRISPR-Cas endonucleases. However, due to their large size and lack of intrinsic localization signals, Cas13 fusion proteins are inefficiently localized to the mammalian nucleus. The EraseR fusion protein is effectively and efficiently delivered to the nucleus allowing for targeted nuclear RNA cleavage and degradation. Thus, EraseR allows for non-genomic manipulation of gene expression, useful for both basic research and therapeutic applications.

[0017] Thus, disclosed herein are compositions and methods for decreasing the number of a nuclear RNA in a subject involving a fusion protein comprising a Cas protein and an NLS.

[0018] The fusion protein may comprise an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 702.

[0019] Also disclosed herein is a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and an NLS.

[0020] The nucleic acid molecule may comprise a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 723.

[0021] Also disclosed herein are novel fusion proteins which allow for specific visualization of nuclear RNA. This fusion protein, termed HilightR herein, comprises a Cas protein and a fluorescent protein. Mutations in Cas13 generates a catytically dead enzyme (dCas) but retains RNA binding affinity. Thus, a fusion of dCas13 and a fluorescent protein allows for targeted visualization of RNA. Accordingly, HilightR allows for visualization of RNA, including nuclear RNA.

[0022] CRISPR-Cas13 systems bind only to RNA and function as specific endoribonucleases to cleave target RNAs, bypassing the risk of germline editing that is associated with DNA-binding CRISPR-Cas endonucleases. However, due to their large size and lack of intrinsic localization signals, Cas13 fusion proteins are inefficiently localized to the mammalian nucleus. The EraseR fusion protein is effectively and efficiently delivered to the nucleus allowing for targeted nuclear RNA cleavage and degradation. Thus, EraseR allows for non-genomic manipulation of gene expression, useful for both basic research and therapeutic applications.

[0023] Disclosed herein is therefore a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a fluorescent protein. The fusion protein may further comprise a nuclear localization signal. The fusion protein may further comprise a linker. The linker may link the Cas protein and fluorescent protein. The fusion protein may comprise a tag.

[0024] The fusion protein may comprise an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 699-701.

[0025] Also disclosed herein is a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a fluorescent protein. The fusion protein may further comprise a nuclear localization signal. The fusion protein may further comprise a linker. The linker may link the Cas protein and fluorescent protein. The fusion protein may comprise a tag.

[0026] A nucleic acid molecule may comprise a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 728-730.Definitions

[0027] Unless defined otherwise, 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.

[0028] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well-known and commonly employed in the art.

[0029] Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.

[0030] The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well-known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.

[0031] The term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0032] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0033] "Antisense" refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0034] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0035] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0036] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0037] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0038] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal or cell whether in vitro or in vivo, amenable to the methods described herein. In one embodiment, the subjects include vertebrates and invertebrates. Invertebrates include, but are not limited to, Drosophila melanogaster and Caenorhabditis elegans. Vertebrates include, but are not limited to, primates, rodents, domestic animals or game animals. Primates include, but are not limited to, chimpanzees, cynomologous monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include, but are not limited to, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., zebrafish, trout, catfish and salmon). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0039] By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0040] In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0041] A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

[0042] A "coding region" of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0043] "Complementary" as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In one embodiment, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0044] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0045] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0046] The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0047] As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0048] The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0049] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0050] The term "isolated" when used in relation to a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences (e.g., a specific mRNA sequence encoding a specific protein), are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).

[0051] The term "isolated" when used in relation to a polypeptide, as in "isolated protein" or "isolated polypeptide" refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated polypeptide is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state they exist in nature.

[0052] By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid" typically refers to large polynucleotides.

[0053] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.

[0054] The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."

[0055] By "expression cassette" is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter / regulatory sequences necessary for transcription and, optionally, translation of the coding sequence.

[0056] The term "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of sequences encoding amino acids in such a manner that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.

[0057] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in an inducible manner.

[0058] 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 are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.

[0059] "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.

[0060] An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds to the promoter is present.

[0061] A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0062] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0063] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0064] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0065] The term "RNA" as used herein is defined as ribonucleic acid.

[0066] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.

[0067] A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

[0068] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0069] "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0070] A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0071] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Fusion Proteins

[0072] In one aspect, the present invention is based on the development of novel fusions of editing proteins which are effectively delivered to the nucleus. In one embodiment, the fusion protein is effectively delivered to the nucleus and is capable of modulating the cleavage and / or polyadenylation of nuclear RNA.EraseR

[0073] Disclosed herein are novel fusions of editing proteins which are effectively delivered to the nucleus. The fusion proteins may comprise an editing protein having a first amino acid sequence and a nuclear localization signal (NLS) having a second amino acid sequence.

[0074] The editing protein may include, but is not limited to, a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN) protein, and a protein having a DNA or RNA binding domain.

[0075] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. The Cas protein may have DNA or RNA cleavage activity. The Cas protein may direct cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The Cas protein may direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In one embodiment, the Cas protein is Cas9, Cas13, or Cpfl. The Cas protein may be catalytically deficient (dCas).

[0076] The Cas protein may be Cas13. The Cas protein may be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, dUrCas13d, or a variant thereof. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0077] The Cas protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:1-48. The Cas protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:1-48. The Cas protein may comprise a sequence of one of SEQ ID NOs: 1-48. The Cas protein may comprise a sequence of one of SEQ ID NOs:1-46.

[0078] The NLS may be a retrotransposon NLS. The NLS may be derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus Ela or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen.

[0079] The NLS may be a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. The Ty1 NLS may comprise an amino acid sequence of SEQ ID NO:75. The Ty2 NLS may comprise an amino acid sequence of SEQ ID NO:76. The MAK11 NLS may comprise an amino acid sequence of SEQ ID NO:77. The NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. The NLS protein may comprise a sequence of one of SEQ ID NOs: 75-695.

[0080] The NLS may be a Tyl-like NLS. For example, the Ty1-like NLS comprises KKRX motif. The Ty1-like NLS may comprise KKRX motif at the N-terminal end. The Ty1-like NLS may comprise KKR motif. The Ty1-like NLS may comprise KKR motif at the C-terminal end. The Tyl-like NLS may comprise a KKRX and a KKR motif. The Ty1-like NLS may comprise a KKRX at the N-terminal end and a KKR motif at the C-terminal end. The Ty1-like NLS may comprise at least 20 amino acids. The Ty1-like NLS may comprise between 20 and 40 amino acids. The Ty1-like NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. The NLS may comprise a sequence of one of SEQ ID NOs: 83-695, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. The Ty1-like NLS protein may comprise a sequence of one of SEQ ID NOs: 83-695.

[0081] The NLS may comprise two copies of the same NLS. For example, the NLS may comprise a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0082] The fusion protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:702. The fusion protein may comprise a sequence of SEQ ID NO: 702.HilightR

[0083] Also disclosed herein are fusions of editing proteins and fluorescent proteins which are effectively delivered to the nucleus. These fusion proteins combine the visualization capability of the fluorescent protein and the programmable DNA targeting capability of catalytically dead Cas. The fusion proteins may comprise a CRISPR-associated (Cas) protein having a first amino acid sequence, and a fluorescent protein having a second amino acid sequence. The fusion protein may comprise a nuclear localization signal having a third amino acid sequence. The fusion protein may comprise a linker having a fourth amino acid sequence. The linker may link the Cas protein and fluorescent protein. The fusion protein may comprise a purification and / or detection tag having a fifth amino acid sequence.

[0084] The editing protein may include, but is not limited to, a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN) protein, and a protein having a DNA or RNA binding domain.

[0085] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. The Cas protein may have DNA or RNA cleavage activity. The Cas protein may direct cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The Cas protein may direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. The Cas protein may be Cas9, Cas13, or Cpfl. The Cas protein may be catalytically deficient (dCas).

[0086] The Cas protein may be Cas13. The Cas protein may be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0087] The Cas protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. The Cas protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. The Cas protein may comprise a sequence of a variant of one of SEQ ID NOs: 1-48, wherein the variant renders the Cas protein catalytically inactive. The Cas protein may comprise a sequence of one of SEQ ID NOs: 1-46 having one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions renders the Cas protein catalytically inactive. The Cas protein may comprise a sequence of one of SEQ ID NOs: 1-48. The Cas protein may comprise a sequence of one of SEQ ID NOs:47-48.

[0088] The NLS may be a retrotransposon NLS. The NLS may be derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen. The NLS may be a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. The Ty1 NLS may comprise an amino acid sequence of SEQ ID NO:75. The Ty2 NLS may comprise an amino acid sequence of SEQ ID NO:76. The MAK11 NLS may comprise an amino acid sequence of SEQ ID NO:77. The NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. The NLS protein may comprise a sequence of one of SEQ ID NOs: 75-695.

[0089] The NLS may be a Ty1-like NLS. For example, The Ty1-like NLS may comprise KKRX motif. The Ty1-like NLS may comprise KKRX motif at the N-terminal end. The Ty1-like NLS may comprise KKR motif. The Ty1-like NLS may comprise KKR motif at the C-terminal end. The Ty1-like NLS may comprise a KKRX and a KKR motif. The Ty1-like NLS may comprise a KKRX at the N-terminal end and a KKR motif at the C-terminal end. The Ty1-like NLS may comprise at least 20 amino acids. The Ty1-like NLS may comprise between 20 and 40 amino acids. The Ty1-like NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. The NLS may comprise a sequence of one of SEQ ID NOs: 83-695, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. The Ty1-like NLS protein may comprise a sequence of one of SEQ ID NOs: 83-695.

[0090] The NLS may comprise two copies of the same NLS. For example, the NLS may comprise a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0091] The fluorescent protein may be eGFP, mCherry, mCherry-MBNL1, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), 7xS11, sfCherry, S11, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, Blue Fluorescent Proteins, EBFP, EBFP2, Azurite, mTagBFP, Cyan Fluorescent Proteins, eCFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Yellow Fluorescent Proteins, EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Orange Fluorescent Proteins, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, Red Fluorescent Proteins, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0092] The fluorescent protein may be eGFP, mCherry, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry, or 7xS11. The fluorescent protein may comprise an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 59-66. The fluorescent protein may comprise an amino acid sequence of one of SEQ ID NOs: 59-66.

[0093] The fusion protein may comprise a purification and / or detection tag. The tag may be on the N-terminal end of the fusion protein. The tag may comprise an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74. The tag may comprise an amino acid sequence of SEQ ID NO: 74.

[0094] The fusion protein may comprise a linker. The linker may link the Cas protein and fluorescent protein. The linker may be connected to the C-terminal end of the Cas protein and to the N-terminal end of the fluorescent protein. The linker may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:67-73. The linker may comprise a sequence at of one of SEQ ID NOs: 67-73.

[0095] The fusion protein may comprise an amino acid sequence 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:699-701. The fusion protein may comprise an amino acid sequence of one of SEQ ID NOs: 699-701.Postscriptr

[0096] In one aspect, the present invention is based on the development of novel fusions of editing proteins and cleavage and / or polyadenylation proteins which are effectively delivered to the nucleus. These fusion proteins combine the catalytic activity of the cleavage and / or polyadenylation protein and the programmable DNA targeting capability of catalytically dead Cas. In one embodiment, the present invention provides fusion proteins comprising a CRISPR-associated (Cas) protein having a first amino acid sequence, and a cleavage and / or polyadenylation protein having a second amino acid sequence wherein the cleavage or polyadenylation protein is NUDT21. In one embodiment, the fusion protein comprises a nuclear localization signal having a third amino acid sequence. In one embodiment, the fusion protein comprises a linker having a fourth amino acid sequence. In one embodiment, the linker links the Cas protein and cleavage and / or polyadenylation protein. In one embodiment, the fusion protein comprises a purification and / or detection tag having a fifth amino acid sequence.

[0097] The editing protein is a CRISPR-associated (Cas) protein.

[0098] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In one embodiment, the Cas protein is Cas9, Cas13, or Cpfl. In one embodiment, Cas protein is catalytically deficient (dCas).

[0099] In one embodiment, Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0100] In one embodiment, the Cas protein comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. In one embodiment, the Cas protein comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. In one embodiment, the Cas protein comprises a sequence of a variant of one of SEQ ID NOs: 1-48, wherein the variant renders the Cas protein catalytically inactive. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 1-46 having one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions renders the Cas protein catalytically inactive. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 1-48. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs:47-48.

[0101] In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen. In one embodiment, the NLS is a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises an amino acid sequence of SEQ ID NO:75. In one embodiment, the Ty2 NLS comprises an amino acid sequence of SEQ ID NO:76. In one embodiment, the MAK11 NLS comprises an amino acid sequence of SEQ ID NO:77. In one embodiment, the NLS comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. In one embodiment, the NLS protein comprises a sequence of one of SEQ ID NOs: 75-695.

[0102] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises KKRX motif. In one embodiment, the Ty1-like NLS comprises KKRX motif at the N-terminal end. In one embodiment, the Ty1-like NLS comprises KKR motif. In one embodiment, the Ty1-like NLS comprises KKR motif at the C-terminal end. In one embodiment, the Ty1-like NLS comprises a KKRX and a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKRX at the N-terminal end and a KKR motif at the C-terminal end. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises between 20 and 40 amino acids. In one embodiment, the Ty1-like NLS comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. In one embodiment, the NLS comprises a sequence of one of SEQ ID NOs: 83-695, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. In one embodiment, the Ty1-like NLS protein comprises a sequence of one of SEQ ID NOs: 83-695.

[0103] In one embodiment, the NLS comprises two copies of the same NLS. For example, in one embodiment, the NLS comprises a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0104] The cleavage or polyadenylation protein is an RNA binding protein of the human 3' end processing machinery. The cleavage or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is human NUDT21, Worm NUDT21, Fly NUDT21, Zebrafish NUDT21 NUDT21_R63S, NUDT21_F103A, or a tandem dimer of NUDT21.

[0105] In one embodiment, the cleavage or polyadenylation protein comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:49-58. In one embodiment, the cleavage and / or polyadenylation protein comprises an amino acid sequence of one of SEQ ID NOs: 49-58. In one embodiment, the cleavage or polyadenylation protein comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 51-58. In one embodiment, the cleavage or polyadenylation protein comprises an amino acid sequence of SEQ ID NO:51-58.

[0106] In one embodiment, the fusion protein comprises a purification and / or detection tag. In one embodiment, the tag is on the N-terminal end of the fusion protein. In one embodiment, the tag comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74. In one embodiment, the tag comprises an amino acid sequence of SEQ ID NO: 74.

[0107] In one embodiment, the fusion protein comprises a linker. In one embodiment, the linker links the Cas protein and cleavage or polyadenylation protein. In one embodiment, the linker is connected to the C-terminal end of the Cas protein and to the N-terminal end of the cleavage or polyadenylation protein. In one embodiment, the linker comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:67-73. In one embodiment, the linker comprises a sequence at of one of SEQ ID NOs: 67-73.

[0108] In one embodiment, the fusion protein comprises an amino acid sequence 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:696-698.In one embodiment, the fusion protein comprises an amino acid sequence of one of SEQ ID NOs:696-698.

[0109] The fusion protein of the present invention may be made using chemical methods. For example, fusion protein can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high-performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0110] The invention should also be construed to include any form of a fusion protein having substantial homology to a fusion-protein disclosed herein. In one embodiment, a fusion protein which is "substantially homologous" is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous, about 95% homologous, or about 99% homologous to amino acid sequence of a fusion-protein disclosed herein.

[0111] The fusion protein may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a fusion protein may be confirmed by amino acid analysis or sequencing.

[0112] The variants of the fusion protein according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the fusion protein of the present invention, (iv) fragments of the peptides and / or (v) one in which the fusion protein is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0113] As known in the art the "similarity" between two fusion proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence. In one embodiment, variants are different from the original sequence in less than 40% of residues per segment of interest different from the original sequence in less than 25% of residues per segment of interest, different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence and / or the ability to stimulate the differentiation of a stem cell into the osteoblast lineage. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences may be determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0114] The fusion protein of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0115] The fusion protein of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0116] A fusion protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0117] Cyclic derivatives of the fusion proteins of the invention are also part of the present invention. Cyclization may allow the fusion protein to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0118] It may be desirable to produce a cyclic fusion protein which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

[0119] The invention also relates to peptides comprising a fusion protein comprising Cas13 and a cleavage or polyadenylation protein, wherein the fusion protein is itself fused to, or integrated into, a target protein, and / or a targeting domain capable of directing the chimeric protein to a desired cellular component or cell type or tissue. The chimeric proteins may also contain additional amino acid sequences or domains. The chimeric proteins are recombinant in the sense that the various components are from different sources, and as such are not found together in nature (i.e., are heterologous).

[0120] In one embodiment, the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence directing the protein to associate with for example vesicles or with the nucleus. In one embodiment, the targeting domain can target a peptide to a particular cell type or tissue. For example, the targeting domain can be a cell surface ligand or an antibody against cell surface antigens of a target tissue. A targeting domain may target the peptide of the invention to a cellular component.

[0121] A peptide of the invention may be synthesized by conventional techniques. For example, the peptides or chimeric proteins may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1, for classical solution synthesis). By way of example, a peptide of the invention may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0122] N-terminal or C-terminal fusion proteins comprising a peptide or chimeric protein of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide or chimeric protein, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the fusion protein fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0123] Peptides of the invention may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to particular proteins. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0124] The peptides and chimeric proteins of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.Nucleic Acids

[0125] In one aspect, the present invention is based on the development of nucleic acids encoding novel fusions of editing proteins which are effectively delivered to the nucleus. In one embodiment, the nucleic acid encodes a fusion protein that can be effectively delivered to the nucleus and is capable of modulating the cleavage and / or polyadenylation of nuclear RNA.EraseR

[0126] Described herein is a nucleic acid molecule encoding a fusion protein. The nucleic acid molecule may comprise a nucleic acid sequence encoding an editing protein; and a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0127] The editing protein may include, but is not limited to, a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN) protein, and a protein having a DNA or RNA binding domain.

[0128] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. The Cas protein may have DNA or RNA cleavage activity. The Cas protein may direct cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The Cas protein may direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. The Cas protein may be Cas9, Cas13, or Cpfl. Cas protein may be catalytically deficient (dCas).

[0129] The Cas protein may be Cas13. The Cas protein may be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0130] The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-48. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs:1-46.

[0131] The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 705-708. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence of one of SEQ ID NOs:705-708. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence of one of SEQ ID NOs:707-708.

[0132] The NLS may be a retrotransposon NLS. The NLS may be derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen.

[0133] The NLS may be a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. The Ty1 NLS may comprise an amino acid sequence of SEQ ID NO:75. The Ty2 NLS may comprise an amino acid sequence of SEQ ID NO:76. The MAK11 NLS may comprise an amino acid sequence of SEQ ID NO:77.

[0134] The NLS may be a Tyl-like NLS. For example, the Ty1-like NLS may comprise KKRX motif. The Ty1-like NLS may comprise KKRX motif at the N-terminal end. The Ty1-like NLS may comprise KKR motif. The Ty1-like NLS may comprise KKR motif at the C-terminal end. The Ty1-like NLS may comprise a KKRX and a KKR motif. The Ty1-like NLS may comprise a KKRX at the N-terminal end and a KKR motif at the C-terminal end. The Ty1-like NLS may comprise at least 20 amino acids. The Ty1-like NLS may comprise between 20 and 40 amino acids. The Ty1-like NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. The NLS may comprise a sequence of one of SEQ ID NOs: 83-695, wherein the sequence may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. The NLS may comprise a sequence of one of SEQ ID NOs: 83-695, wherein the sequence may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. The Ty1-like NLS protein may comprise a sequence of one of SEQ ID NOs: 83-695.

[0135] The NLS may comprise two copies of the same NLS. For example, the NLS may comprise a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0136] The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 75-695. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:724. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence of SEQ ID NO:724.

[0137] The nucleic acid molecule encoding a fusion protein may comprise a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:702. The fusion protein may comprise a sequence of SEQ ID NO: 702. The nucleic acid molecule encoding a fusion protein may comprise a sequence encoding an amino acid sequence of SEQ ID NO: 702.HilightR

[0138] Disclosed herein are novel nucleic acids encoding fusion proteins comprising an editing protein and a fluorescent protein which are effectively delivered to the nucleus. The nucleic acid molecule may comprise a nucleic acid sequence encoding an editing protein; and a nucleic acid sequence encoding a fluorescent protein. The nucleic acid molecule may comprise a nucleic acid sequence encoding a nuclear localization signal (NLS). The nucleic acid molecule may comprise a nucleic acid sequence encoding a linker. The nucleic acid molecule may comprise a nucleic acid sequence encoding a tag.

[0139] The editing protein may include, but is not limited to, a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN) protein, and a protein having a DNA or RNA binding domain.

[0140] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. The Cas protein may have DNA or RNA cleavage activity. The Cas protein may direct cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The Cas protein may direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. The Cas protein may be Cas9, Cas13, or Cpfl. The Cas protein may be catalytically deficient (dCas).

[0141] The Cas protein may be Cas13. The Cas protein may be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0142] The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of a variant of one of SEQ ID NOs: 1-48, wherein the variant renders the Cas protein catalytically inactive. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-46 having one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions renders the Cas protein catalytically inactive. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs:1-48. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs:47-48.

[0143] The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 705-708. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence of one of SEQ ID NOs:705-708. The nucleic acid sequence encoding a Cas protein may comprise a nucleic acid sequence of one of SEQ ID NOs:707-708.

[0144] The NLS may be a retrotransposon NLS. The NLS may be derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen.

[0145] The NLS may be a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. The Ty1 NLS may comprise an amino acid sequence of SEQ ID NO:75. The Ty2 NLS may comprise an amino acid sequence of SEQ ID NO:76. The MAK11 NLS may comprise an amino acid sequence of SEQ ID NO:77.

[0146] The NLS may be a Tyl-like NLS. For example, the Ty1-like NLS may comprise KKRX motif. The Ty1-like NLS may comprise KKRX motif at the N-terminal end. The Ty1-like NLS may comprise KKR motif. The Ty1-like NLS may comprise KKR motif at the C-terminal end. The Ty1-like NLS may comprise a KKRX and a KKR motif. The Ty1-like NLS may comprise a KKRX at the N-terminal end and a KKR motif at the C-terminal end. The Ty1-like NLS may comprise at least 20 amino acids. The Ty1-like NLS may comprise between 20 and 40 amino acids. The Ty1-like NLS may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. The NLS may comprise a sequence of one of SEQ ID NOs: 83-695, wherein the sequence may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. The Ty1-like NLS protein may comprise a sequence of one of SEQ ID NOs: 83-695.

[0147] The NLS may comprise two copies of the same NLS. For example, the NLS may comprise a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0148] The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 75-695. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:724. The nucleic acid sequence encoding a NLS may comprise a nucleic acid sequence of SEQ ID NO:724.

[0149] The fluorescent protein may be eGFP, mCherry, mCherry-MBNL1, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry 7xS11, S11, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, Blue Fluorescent Proteins, EBFP, EBFP2, Azurite, mTagBFP, Cyan Fluorescent Proteins, eCFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Yellow Fluorescent Proteins, EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Orange Fluorescent Proteins, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, Red Fluorescent Proteins, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0150] The fluorescent protein may be eGFP, mCherry, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry or 7xS11. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:59-66. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence of one of SEQ ID NOs: 59-66.

[0151] The nucleic acid molecule may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 718-721. The nucleic acid molecule may comprise a sequence of one of SEQ ID NOs: 718-721.

[0152] The nucleic acid molecule may comprise a sequence encoding a tag. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence of SEQ ID NO: 7474.

[0153] The nucleic acid molecule may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:723. The nucleic acid molecule may comprise a sequence of SEQ ID NO: 723.

[0154] The nucleic acid molecule may comprise a sequence encoding a linker. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 67-73. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence of one of SEQ ID NOs: 67-73.74

[0155] The nucleic acid molecule may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:722. The nucleic acid molecule may comprise a sequence of SEQ ID NO: 722723.

[0156] The nucleic acid molecule encoding a fusion protein may comprise a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 699-701. The nucleic acid molecule encoding a fusion protein may comprise a sequence encoding an amino acid sequence of one of SEQ ID NOs: 699-701.

[0157] The nucleic acid molecule encoding a fusion protein may comprise a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:728-730. The nucleic acid molecule encoding a fusion protein may comprise a sequence of one of SEQ ID NOs:728-730.PostscriptR

[0158] The present invention is based on the development of novel nucleic acids encoding fusion proteins comprising an editing protein and a cleavage or polyadenylation protein which are effectively delivered to the nucleus, wherein the cleavage or polyadenylation protein is NUDT21. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an editing protein; and a nucleic acid sequence encoding a cleavage and / or polyadenylation wherein the cleavage or polyadenylation protein is NUDT21. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a nuclear localization signal (NLS). In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a linker. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a tag.

[0159] The editing protein is a CRISPR-associated (Cas) protein.

[0160] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpfl, LbCpfl, FnCpfl, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In one embodiment, the Cas protein is Cas9, Cas13, or Cpf1. In one embodiment, Cas protein is catalytically deficient (dCas).

[0161] In one embodiment, Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RerCas13a, ResCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3).

[0162] In one embodiment, the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:1-48. In one embodiment, the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of a variant of one of SEQ ID NOs: 1-48, wherein the variant renders the Cas protein catalytically inactive. the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-46 having one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions renders the Cas protein catalytically inactive. the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-48 the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs:47-48.

[0163] In one embodiment, the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 705-708. In one embodiment, the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs:705-708. In one embodiment, the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs:707-708.

[0164] In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 ("SV40") T-antigen.

[0165] In one embodiment, the NLS is a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises an amino acid sequence of SEQ ID NO:75. In one embodiment, the Ty2 NLS comprises an amino acid sequence of SEQ ID NO:76. In one embodiment, the MAK11 NLS comprises an amino acid sequence of SEQ ID NO:77.

[0166] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises KKRX motif. In one embodiment, the Ty1-like NLS comprises KKRX motif at the N-terminal end. In one embodiment, the Ty1-like NLS comprises KKR motif. In one embodiment, the Ty1-like NLS comprises KKR motif at the C-terminal end. In one embodiment, the Ty1-like NLS comprises a KKRX and a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKRX at the N-terminal end and a KKR motif at the C-terminal end. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises between 20 and 40 amino acids. In one embodiment, the Ty1-like NLS comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:83-695. In one embodiment, the NLS comprises a sequence of one of SEQ ID NOs: 83-695, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, insertions, deletions or substitutions. In one embodiment, the Ty1-like NLS protein comprises a sequence of one of SEQ ID NOs: 83-695.

[0167] In one embodiment, the NLS comprises two copies of the same NLS. For example, in one embodiment, the NLS comprises a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0168] In one embodiment, the nucleic acid sequence encoding a NLS comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:75-695. In one embodiment, the nucleic acid sequence encoding a NLS comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding a NLS comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:724. In one embodiment, the nucleic acid sequence encoding a NLS comprises a nucleic acid sequence of SEQ ID NO:724.

[0169] In one embodiment, the cleavage or polyadenylation protein is an RNA binding protein of the human 3' end processing machinery. The cleavage or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is human NUDT21, Worm NUDT21, Fly NUDT21, Zebrafish NUDT21, NUDT21_R63S, NUDT21_F103A, or a tandem dimer of NUDT21.

[0170] In one embodiment, nucleic acid molecule comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-58. In one embodiment, nucleic acid molecule comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 49-58.

[0171] In one embodiment, nucleic acid molecule comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 51-58. In one embodiment, nucleic acid molecule comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 51-58.

[0172] In one embodiment, nucleic acid molecule comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 711-717. In one embodiment, nucleic acid molecule comprises a sequence of one of SEQ ID NOs: 711709-717.

[0173] In one embodiment, nucleic acid molecule comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 709-717. In one embodiment, nucleic acid molecule comprises a sequence of one of SEQ ID NOs: 709-717.

[0174] In one embodiment, the nucleic acid molecule comprises a sequence encoding a tag. In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:74. In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence of SEQ ID NO: 7474.

[0175] In one embodiment, nucleic acid molecule comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:723. In one embodiment, nucleic acid molecule comprises a sequence of SEQ ID NO: 723.

[0176] In one embodiment, the nucleic acid molecule comprises a sequence encoding a linker. In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 67-73. In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 67-73.74

[0177] In one embodiment, nucleic acid molecule comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:722. In one embodiment, nucleic acid molecule comprises a sequence of SEQ ID NO: 722723.

[0178] In one embodiment, the nucleic acid molecule encoding a fusion protein comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698. In one embodiment, the nucleic acid molecule encoding a fusion protein comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 696-698.

[0179] In one embodiment, the nucleic acid molecule encoding a fusion protein comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 725-727. In one embodiment, the nucleic acid molecule encoding a fusion protein comprises a sequence of one of SEQ ID NOs:725-727.

[0180] The isolated nucleic acid sequence encoding a fusion protein can be obtained using any of the many recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0181] The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a fusion protein of the invention. In one embodiment, the composition comprises an isolated RNA molecule encoding a fusion protein of the invention, or a functional fragment thereof.

[0182] The nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and / or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention. For example, in order to enhance the stability, the 3'-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect function of the molecule.

[0183] In one embodiment of the present invention the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

[0184] Non-limiting examples of nucleotide analogues include sugar- and / or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or ON, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0185] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

[0186] In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modification of one or more nucleotides. In certain embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.

[0187] In one embodiment, the nucleic acid molecule includes a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0188] In certain embodiments, the nucleic acid molecule of the invention has one or more of the following properties: Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, or as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, or different from that which occurs in the human body. While they are referred to as "modified RNAs" they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

[0189] Modifications of the nucleic acid of the invention may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0190] Also disclosed herein is a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.

[0191] In brief summary, the expression of natural or synthetic nucleic acids encoding a fusion protein of the invention is typically achieved by operably linking a nucleic acid encoding the fusion protein of the invention or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0192] The vectors may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. The vector may be a gene therapy vector.

[0193] The isolated nucleic acid of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0194] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).Delivery Systems and Methods

[0195] In one aspect, the invention relates to the development of novel lentiviral packaging and delivery systems for use in a method of modulating the cleavage, polyadenylation or both of an RNA transcript. The lentiviral particle delivers the viral enzymes as proteins. In this fashion, lentiviral enzymes are short lived, thus limiting the potential for off-target editing due to long term expression though the entire life of the cell. The incorporation of editing components, or traditional CRISPR-Cas editing components as proteins in lentiviral particles is advantageous, given that their required activity is only required for a short period of time. Thus, in one embodiment, the invention provides a lentiviral delivery system and methods of delivering the compositions of the invention, editing genetic material, and nucleic acid delivery using lentiviral delivery systems.

[0196] In one aspect, the delivery system comprises (1) a packaging plasmid (3) an envelope plasmid, and (4) a VPR plasmid. In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises the D116N integrase mutation.

[0197] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g-protein (VSV-g) envelope protein. In one embodiment, the envelope protein can be selected based on the desired cell type.

[0198] In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein and a cleavage or polyadenylation protein. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein and a cleavage or polyadenylation protein. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein and a cleavage or polyadenylation protein. In one embodiment, the fusion protein comprises a protease cleavage site between VPR and the Cas protein cleavage or polyadenylation protein. In one embodiment, the VPR plasmid packaging plasmid further comprises a sequence encoding a guide RNA sequence.

[0199] In one embodiment, the packaging plasmid, transfer plasmid, envelope plasmid, and VPR plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein. In one embodiment, the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein. In one embodiment, the cell transcribes and translates the fusion protein to produce the VPR-fusion protein. In one embodiment, the cell transcribes and translates the fusion protein to produce the VPR-fusion protein. In one embodiment, the cell transcribes the nucleic acid sequence encoding the guide RNA.

[0200] In one embodiment, the gag-pol protein, envelope polyprotein, and VPR-fusion protein, which is bound to the guide RNA, are packaged into a viral particle. In one embodiment, the viral particles are collected from the cell media. In one embodiment, VPR is cleaved from the fusion protein in the viral particle via the protease site to provide a Cas-fusion protein. In one embodiment, the viral particles transduce a target cell, wherein the guide RNA binds a target region of an RNA thereby targeting the Cas fusion protein.

[0201] Further, a number of additional viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0202] For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0203] In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). The term "AAV vector" means a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, and AAV-9. AAV vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0204] AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Despite the high degree of homology, the different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Since most of the studies have been done with pseudotyped vectors in which the vector DNA flanked with AAV2 ITR is packaged into capsids of alternate serotypes, it is clear that the biological differences are related to the capsid rather than to the genomes. Recent evidence indicates that DNA expression cassettes packaged in AAV 1 capsids are at least 1 log 10 more efficient at transducing cardiomyocytes than those packaged in AAV2 capsids. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV 1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).

[0205] Desirable AAV fragments for assembly into vectors include the cap proteins, including the vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Thus exemplary AAVs, or artificial AAVs, suitable for expression of one or more proteins, include AAV2 / 8 (see U.S. Pat. No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication No. WO2005 / 033321), AAV2 / 6 (U.S. Pat. No. 6,156,303), and AAVrh8 (International Patent Publication No. WO2003 / 042397), among others.

[0206] In certain embodiments, the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0207] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0208] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0209] Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

[0210] In order to assess the expression of a fusion protein of the invention, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0211] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0212] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0213] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An exemplary method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0214] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0215] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0216] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0217] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0218] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.Systems

[0219] In one aspect, the present invention provides a system for modulating the cleavage, polyadenylation or both of an RNA transcript. In one embodiment the system comprises, in one or more vectors, a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a CRISPR-associated (Cas) protein, a cleavage and / or polyadenylation protein wherein the cleavage or polyadenylation protein is NUDT21, and a nuclear localization signal (NLS); and a nucleic acid sequence coding a CRISPR-Cas system crRNA. In one embodiment, the CRISPR-Cas system crRNA substantially hybridizes to a target RNA sequence in the RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA are in the same vector. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA are in different vectors.

[0220] In one embodiment, the nucleic acid sequence encoding a fusion protein comprises (1) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-46; (2) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 51-58; and (3) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 75-695 . In one embodiment, the nucleic acid sequence encoding a fusion protein comprises nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698. In one embodiment, the nucleic acid sequence encoding a fusion protein comprises (1) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 1-48; (2) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 51-58; and (3) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding a fusion protein comprises nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 696-698.

[0221] Also disclosed herein is a system for decreasing the number of an RNA transcript in a subject comprising, in one or more vectors, a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a CRISPR-associated (Cas) protein and a nuclear localization signal (NLS); and a nucleic acid sequence coding a CRISPR-Cas system crRNA. The CRISPR-Cas system crRNA may substantially hybridize to a target RNA sequence in the RNA transcript. The nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA may be in the same vector. The nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA may be in different vectors.

[0222] The nucleic acid sequence encoding a fusion protein may comprise (1) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-46; and (2) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 75-695 . The nucleic acid sequence encoding a fusion protein may comprise nucleic acid sequence encoding an amino acid at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 702. The nucleic acid sequence encoding a fusion protein may comprise (1) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs:1-48; and (2) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 75-695. The nucleic acid sequence encoding a fusion protein may comprise nucleic acid sequence encoding an amino acid of SEQ ID NO: 702.

[0223] Disclosed herein is a system for visualizing nuclear RNA in a subject. The system may comprise, in one or more vectors, a nucleic acid sequence encoding a fusion protein, wherein the fusion protein may comprise a CRISPR-associated (Cas) protein, a fluorescent protein, and a nuclear localization signal (NLS); and a nucleic acid sequence coding a CRISPR-Cas system crRNA. The CRISPR-Cas system crRNA may substantially hybridize to a target RNA sequence in the RNA transcript. The nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA may be in the same vector. The nucleic acid sequence encoding the fusion protein and the nucleic acid sequence coding a CRISPR-Cas system crRNA may be in different vectors.

[0224] The nucleic acid sequence encoding a fusion protein may comprise (1) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-46; (2) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 59-66; and (3) a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 699-701. nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of The nucleic acid sequence encoding a fusion protein may comprise (1) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 1-48; (2) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 59-66; and (3) nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 75-695. The nucleic acid sequence encoding a fusion protein may comprise nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 699-701.

[0225] The systems and vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR 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, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector systems can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0226] Vectors may be introduced and propagated in a prokaryote. 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.

[0227] 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., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0228] 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.).

[0229] 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).

[0230] 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., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0231] 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) 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).

[0232] In some embodiments, a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system. In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli(Ishino et al., J. Bacteriol., 169:5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171:3553-3556

[1989] ), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10: 1057-1065

[1993] ; Hoe et al., Emerg. Infect. Dis., 5:254-263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] ; and Mojica et al., Mol. Microbiol., 36:244-246

[2000] ). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al.,

[2000] , supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0233] As used herein, a "target sequence" refers to a sequence to which a crRNA sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.

[0234] The ability of a crRNA to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A crRNA sequence, and hence a nucleic acid-targeting crRNA may be selected to target any target nucleic acid sequence.

[0235] The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA). In some preferred embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0236] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0237] In some embodiments, a crRNA is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0238] In certain embodiments, a crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, the crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., 5') from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') from the guide sequence or spacer sequence.

[0239] In certain embodiments, the crRNA comprises a stem loop. In one embodiment, the crRNA comprises a single stem loop. In certain embodiments, the direct repeat sequence forms a stem loop. In one embodiment, the direct repeat sequence forms a single stem loop.

[0240] In one embodiment, the crRNA enhances the activity of Cas13 targeting to a target sequence, Cas13 catalytic activity, or both. For example, in one embodiment, the crRNA the crRNA sequence comprises a mutation in its direct repeat sequence. For example, in one embodiment, the crRNA comprises a T17C point mutation. In one embodiment, the crRNA comprises a T18C point mutation.Pharmaceutical Compositions and Formulations

[0241] The invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention. Such a pharmaceutical composition may consist of at least one modulator (e.g., inhibitor or activator) composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one modulator (e.g., inhibitor or activator) composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0242] In an embodiment, the pharmaceutical compositions of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, the pharmaceutical compositions of the invention may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day.

[0243] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0244] Pharmaceutical compositions of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. A composition of the invention may be directly administered to the skin, or any other tissue of a mammal. Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.

[0245] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0246] As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0247] In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0248] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.

[0249] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0250] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA).

[0251] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. An exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0252] In one embodiment, the composition includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the range of about 0.01% to 0.3% and BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. In one embodiment, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%. In some embodiments, the chelating agent is in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidants and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0253] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0254] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0255] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0256] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0257] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0258] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0259] Administration of the compositions of the present invention to a subject, include a mammal, for example a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0260] The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0261] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0262] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0263] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease in a subject.

[0264] In one embodiment, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.

[0265] Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.

[0266] In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., a drug used for treating the same or another disease as that treated by the compositions of the invention) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0267] In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0268] The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.

[0269] Routes of administration of any of the compositions of the invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, and (intra)nasal,), intravesical, intraduodenal, intragastrical, rectal, intra-peritoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, or administration.

[0270] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.Methods of Modulating RNA Cleavage and / or Polyadenylation & Methods of Treatment

[0271] In one aspect, the invention provides in vitro methods of modulating the cleavage, polyadenylation or both of an RNA transcript in a subject. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and a cleavage and / or polyadenylation protein or a fusion protein of the invention comprising a Cas protein a cleavage and / or polyadenylation protein, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript.

[0272] In one embodiment, the subject is a cell.

[0273] According to another aspect of the invention, there is provided a fusion protein or a nucleic acid molecule as discussed above and a guide nucleic acid comprising a sequence complimentary to a target RNA sequence in the RNA transcript for use in in vivo modulation of the cleavage, polyadenylation or both of an RNA transcript in a subject. In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, fruit fly, or roundworm.

[0274] In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 725-727 and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0275] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0276] In one aspect, the invention provides a composition for use in a method of treating a disease or disorder associated with abnormal cleavage and / or polyadenylation. In one embodiment, the composition comprises (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein a cleavage and / or polyadenylation protein, and (2) a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0277] In one embodiment, the composition comprises (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein a cleavage and / or polyadenylation protein and (2) a guide nucleic acid molecule comprising a crRNA targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0278] In one embodiment, the gene is associated with abnormal 3'UTR lengthening, 3'UTR shortening, polyadenylation signal alteration, or other abnormal polyadenylation which causes the disease or disorder. Exemplary diseases or disorders and corresponding targets include, but are not limited to those listed in Table 1.

[0279] For example, in one embodiment, treating a Alzheimer's Disease, wherein the composition comprises (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein a cleavage and / or polyadenylation protein and (2) a guide nucleic acid molecule comprising a crRNA targeting nucleotide sequence complimentary to a target region in a gene selected from the group consisting of ABCA3, EIF2B3, MSTO2P, OGDHL, PARP6, SLC33A1, SUPV3L1, TAF3, WASH7P, JAK1, ABCA3, UBR1, ALDOC, C10ord10, GABARAPL2, KAT2A, POLR3A, SH3BGRL2, TIMM23, TMC6, UNC80, and WTAP. Table 1. Diseases or disorders associated with abnormal cleavage and / or polyadenylation and target gene(s),ConditionTargetDescriptionAlzheimer's diseaseABCA3, EIF2B3, MSTO2P, OGDHL, PARP6, SLC33A1, SUPV3L1, TAF3, WASH7P, JAK1, ABCA3, UBR1, ALDOC, C10ord10, GABARAPL2, KAT2A, POLR3A, SH3BGRL2, TIMM23, TMC6, UNC80, WTAP CCDC92, CRYAB, MAP7D2, PRPH, RTN4, SEC22B, SNAP25, UCHL1, YWHAB, C14ord2, C6orf203, ALDOC, ARL6IP1, CSDE1, CMC2, GAP43, LDHB, MLLTI11, NCL, PFN2, TMODI1, VAMP13'UTR lengthening, and / or 3'UTR shorteningAmyotrophic Lateral Sclerosis3'UTR lengthening, and / or 3'UTR shorteningB-cell differentiationIGHMCSTF2 leads to proximal polyadenylation signal usageCancer, ColorectalDMKN, PDXK, PPIE3'UTR shortening has occurred during tumorigenesisCancer, VariousTP53Polyadenylation signal alteration (AATAAA to AATGAA); Shortening of 3' untranslated regionChronic lymphocytic leukaemiaIntronic lociWidespread polyadenylation at intronic loci inactivates tumor suppressor genesDiabetic nephropathyHGRG-14High-glucose level leads to distal polyadenylation signal usageFragile X SyndromeFMRI1Polymorphic CGG repeat resulting in CpG methylation of the DNA in both the promoter region of the FMR1 gene, and of the expanded repeatFriedreich's AtaxiaYSH1Missense mutations in Yshl of the mRNA cleavage and polyadenylation complex induce (GAA) n repeat expansionsGlioblastomaCCND1Knockdown of CPSF5 induces 3'UTR shorteningIPEX SyndromeFOXP3Polyadenylation signal alteration (AATAAA to AATGAA)Myotonic Dystrophy Type IDMPKExpansion of a CTG repeat in the 3' untranslated region of the DMPK geneMyotonic Dystrophy Type IIZFN9Expansion of a CCTG repeat in the first intron of ZFN9Neonatal diabetesINSDisruptive alteration in polyadenylation signalOculopharyngeal muscular dystrophyPABPN1(GCG) n trinucleotide repeat expansionParkinson's diseaseSNCAParkinson's disease risk factor induces shorter isoformProliferative conditionsRBX1Hyper-activated mTOR leads to usage of proximal polyadenylation signalsSpinocerebellar Ataxia 1ATXN1Polyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 10ATXN10Polyadenylation signal alteration (ATTCT repeat)Spinocerebellar Ataxia 17TATA-box binding proteinPolyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 2ATXN2Polyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 3ATXN3Polyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 31BEAN1Polyadenylation signal alteration (TGGAA repeat)Spinocerebellar Ataxia 6CACNA1APolyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 7TPP1Polyadenylation signal alteration (CAG repeat)Spinocerebellar Ataxia 8ATXN8OSPolyadenylation signal alteration (CTG / CAG repeat)SteroidogenesisSTARBr-cAMP stimulates distal polyadenylation signal usageSystemic lupus erythematosusGIMAP5Polyadenylation signal alteration (AATAAA to AATAGA)Systemic lupus erythematosusIRF5Polyadenylation signal alteration (AATGAA to AATAAA)T-cell activationNF-ATC1Upregulation of CSTF2 stimulates 3'UTR shortening during T-cells activationThrombophiliaF2CG-to-CA variantType I diabetesGIMAP5Polyadenylation signal alteration (AATAAA to AATAGA)Type II diabetesTCF7L2Increased different isoforms by usage of intronic polyadenylation signalsWiskott-Aldrich SyndromeWASLocus alteration resulting in expression of 3' isoform of WAS mRNAα-ThalassaemiaHBA1, HBA2Polyadenylation signal alteration (AATAAA to AATAAG)β-ThalassaemiaHBBPolyadenylation signal alteration (AATAAA to AACAAA; AATAAA to A-; AATAAA to AATAAG) Methods of Decreasing RNA & Methods of Treatment

[0280] Also disclosed herein are methods of decreasing the number of a nuclear RNA in a subject. The nuclear RNA may be abnormal nuclear RNA. The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA.

[0281] The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:702 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:731 and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0282] The method may comprise administering to the subject a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:702 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0283] The subject may be a cell. The cell may be a prokaryotic cell or eukaryotic cell. The cell may be a eukaryotic cell. The cell may be a plants, animals, or fungi cell. The cell may be a plant cell. The cell may be an animal cell. The cell may be a yeast cell.

[0284] The subject may be a mammal. For example, the subject may be a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse. The subject may be a non-mammalian subject. For example, the subject may be a zebrafish, fruit fly, or roundworm.

[0285] The amount of nuclear RNA may be reduced in vitro. The amount of nuclear RNA may be reduced in vivo.

[0286] The nuclear RNA may be nuclear RNA foci. The guide nucleic acid may comprise a sequence complementary to a CTG repeat expansion in the 3'UTR of the human dystrophia myotonica-protein kinase (DMPK) gene. The guide nucleic acid may comprise a sequence of one of SEQ ID NOs:762-764.

[0287] Disclosed herein are methods of treating a subject with a disease or disorder associated with abnormal nuclear RNA. The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA.

[0288] The disease or disorder may be associated with abnormal nuclear RNA. The disease or disorder may be selected from the group consisting of Myotonic Dystrophy type 2 (DM2), Amyotrophic lateral sclerosis (ALS), Huntington's disease-like 2 (HDL2), Spinocerebellar ataxias 8, 31 and 10 (SCA8, -31, -10) and fragile X-associated tremor ataxia syndrome (FXTAS).

[0289] The abnormal nuclear RNA may be toxic nuclear RNA foci. The disease or disorder may be associated with toxic nuclear RNA foci Myotonic Dystrophy type 1. The targeting nucleotide sequence may comprise a sequence complementary to a CTG repeat expansion in the 3'UTR of the human dystrophia myotonica-protein kinase (DMPK) gene. The targeting nucleotide sequence may comprise a sequence selected from the group consisting of SEQ ID NOs:762-764.

[0290] Disclosed herein are methods of cleaving of nuclear RNA in a subject. The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA.

[0291] Disclosed herein are methods of treating a disease or disorder associated with increased gene expression. The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene. The Cas protein may cleave the RNA transcript thereby preventing translation and protein expression.

[0292] Disclosed herein are methods of treating a disease or disorder associated with RNA. For example, the invention provides a method of treating an RNA virus infection. The invention provides a method of treating a DNA virus infection. The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the viral RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the viral RNA. The Cas protein may cleave the RNA transcript thereby preventing translation and expression of viral protein.Methods of Visualizing RNA

[0293] Also disclosed herein are methods of visualizing nuclear RNA in a subject. The method may comprise (A) administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and a fluorescent protein or a fusion protein of the invention comprising a Cas protein and a fluorescent protein, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA; and (B) visualizing the nuclear RNA. In one embodiment, visualizing the RNA occurs via imaging. The Cas protein may bind the guide nucleic acid, the guide nucleic acid may bind to the target RNA sequence and the fluorescent protein may be detected.

[0294] The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 699-701 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 728-730 and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0295] The method may comprise administering to the subject a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 699-701 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0296] The subject may be a cell. The cell may be a prokaryotic cell or eukaryotic cell. The cell may be a eukaryotic cell. The cell may be a plants, animals, or fungi cell. The cell may be a plant cell. The cell may be an animal cell. The cell may be a yeast cell.

[0297] The subject may be a mammal. For example, the subject may be a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse. The subject may be a non-mammalian subject. For example, the subject may be a zebrafish, fruit fly, or roundworm.

[0298] The nuclear RNA may be visualized in vitro. The nuclear RNA may be visualized in vivo.

[0299] The nuclear RNA may be nuclear RNA foci. The crRNA may comprise a sequence complementary to a CTG repeat expansion in the 3'UTR of the human dystrophia myotonica-protein kinase (DMPK) gene. The crNA may comprise a sequence of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0300] Also disclosed herein is a method of diagnosing a disease or disorder associated with abnormal nuclear RNA. The method may comprise administering (A) administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and a fluorescent protein or a fusion protein of the invention comprising a Cas protein and a fluorescent protein, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the abnormal nuclear RNA; (B) visualizing the abnormal nuclear RNA; and (C) diagnosing the disease or disorder when the abnormal nuclear RNA is present.Methods of Treatment and Use

[0301] The present invention provides a composition for use in methods of treating, reducing the symptoms of, and / or reducing the risk of developing a disease or disorder in a subject. For example, in one embodiment, compositions of the invention of treat, reduce the symptoms of, and / or reduce the risk of developing a disease or disorder in a mammal. In one embodiment, the compositions of the invention of treat, reduce the symptoms of, and / or reduce the risk of developing a disease or disorder in a plant. In one embodiment, the compositions of the invention of treat, reduce the symptoms of, and / or reduce the risk of developing a disease or disorder in a yeast organism.

[0302] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic cell or eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plants, animals, or fungi cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is an animal cell. In one embodiment, the cell is a yeast cell.

[0303] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, fruit fly, or roundworm.

[0304] In one embodiment, the disease or disorder is caused by one or more mutations in a genomic locus. Thus, in one embodiment, the disease or disorder is may be treated, reduced, or the risk can be reduced via an element that prevents or reduces mRNA transcript, or prevents or reduces translation of the protein. Thus, in one embodiment, the composition manipulates an RNA transcript.

[0305] In one embodiment, the disease or disorder is caused by abnormal RNA. Thus, in one embodiment, the disease or disorder is may be treated, reduced, or the risk can be reduced via an element that prevents or reduces RNA transcript. Thus, in one embodiment, the composition manipulates an RNA transcript.

[0306] In one embodiment, the composition comprises (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein a cleavage and / or polyadenylation protein, and (2) a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. In one embodiment, the cleavage and / or polyadenylation protein modulates the cleavage and / or polyadenylation of the RNA transcript.

[0307] In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 725-727 and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0308] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 696-698 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0309] Also disclosed herein is a method comprising administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA.

[0310] The method may comprise administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the invention comprising a Cas protein and an NLS or a fusion protein of the invention comprising a Cas protein and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the nuclear RNA. The Cas protein may cleave the RNA transcript.

[0311] The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:702 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript. The method may comprise administering to the subject a nucleic acid molecule comprising a nucleic acid sequence least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:731 and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0312] The method may comprise administering to the subject a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:702 and a guide nucleic acid comprising a targeting nucleotide sequence complimentary to a target region in a gene, wherein the gene encodes the RNA transcript.

[0313] In one embodiment, the disease or disorder is associated with RNA. For example, in one embodiment, the diseases or disorder is an RNA virus or DNA virus infection. In one embodiment, the RNA virus is a positive strand RNA virus, a negative strand RNA virus or a double-stranded RNA virus.

[0314] Exemplary RNA viruses include, but are not limited to, Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2, Primate T-lymphotropic virus 3, Human immunodeficiency virus 1, Human immunodeficiency virus 2, Simian foamy virus, Human picobirnavirus, Colorado tick fever virus, Changuinola virus, Great Island virus, Lebombo virus, Orungo virus, Rotavirus A, Rotavirus B, Rotavirus C, Banna virus, Borna disease virus, Lake Victoria Marburgvirus, Reston ebolavirus, Sudan ebolavirus, Tai forest ebolavirus, Zaire virus, Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus, Newcastle disease virus, Human parainfluenza virus 1, Human parainfluenza virus 3, Hendra virus, Nipah virus, Measles virus, Human respiratory syncytial virus, Human metapneumovirus, Chandipura virus, Isfahan virus, Piry virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis Indiana virus, Vesicular stomatitis New Jersey virus, Australian bat lyssavirus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Mokola virus, Rabies virus, Guanarito virus, Junin virus, Lassa virus, Lymphocytic choriomeningitis virus, Machupo virus, Pichinde virus, Sabia virus, Whitewater Arroyo virus, Bunyamwera virus, Bwamba virus, California encephalitis virus, Caraparu virus, Catu virus, Guama virus, Guaroa virus, Kairi virus, Marituba virus, Oriboca virus, Oropouche virus, Shuni virus, Tacaiuma virus, Wyeomyia virus, Andes virus, Bayou virus, Black creek canal virus, Dobrava-Belgrade virus, Hantaan virus, Laguna Negra virus, New York virus, Puumala virus, Seoul virus, Sin Nombre virus, Crimean-Congo haemorrhagic fever virus, Dugbe virus, Candiru virus, Punta Toro virus, Rift Valley fever virus, Sandfly fever Naples virus, Influenza A virus, Influenza B virus, Influenza C virus, Dhori virus, Thogoto virus, Hepatitis delta virus, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1, Human coronavirus OC43, SARS coronavirus, Human torovirus, Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Encephalomyocarditis virus, Theilovirus, Equine rhinitis A virus, Foot and mouth disease virus, Hepatitis A virus, Human parechovirus, Ljungan virus, Aichi virus, Human astrovirus, Human astrovirus 2, Human astrovirus 3, Human astrovirus 4, Human astrovirus 5, Human astrovirus 6, Human astrovirus 7, Human astrovirus 8, Norwalk virus, Sapporo virus, Aroa virus, Banzi virus, Dengue virus, Ilheus virus, Japanese encephalitis virus, Kokobera virus, Kyasanur forest disease virus, Louping ill virus, Murray Valley encephalitis virus, Ntaya virus, Omsk haemorrhagic fever virus, Powassan virus, Rio Bravo virus, St Louis encephalitis virus, Tick-borne encephalitis virus, Usutu virus, Wesselsbron virus, West Nile virus, Yellow fever virus, Zika virus, Hepatitis C virus, Hepatitis E virus, Barmah Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Getah virus, Mayaro virus, Mucambo virus, O'nyong-nyong virus, Pixuna virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Whataroa virus, Rubella virus.

[0315] In one embodiment, the DNA virus is a single-stranded DNA virus, or a double-stranded DNA virus. Exempary DNA viruses include, but are not lmited to, AAV9, AAV12, AAV18, AAV31, HBV, EBV, KSHV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV45, Merkel cell polymavirus, and MCV.

[0316] In one embodiment, the disease or disorder is associated with abnormal polyadenylation or abnormal RNA. In one embodiment, the method treats the disease or disorder associated with abnormal abnormal polyadenylation or abnormal RNA, wherein the abnormal polyadenylation or abnormal RNA is targeted.

[0317] In one embodiment, the disease or disorder is an endocrine disease. For example, in one embodiment, endocrine diseases include but are not limited to, β-thalassemias, neonatal diabetes,

[0318] IPEX syndrome, Mayer-Rokitanski-Küster-Hausersyndrome, Hypothalamic-pituitary-adrenal axis dysregulation, Adrenal dysfunction, Gonadal dysfunction, Ectopic Cushing syndrome, Pre-eclampsia, Diabetic nephropathy, Type I diabetes, Type II diabetes, and IGF-1 deficiency.

[0319] In one embodiment, the disease or disorder is a tumorigenic disease. For example, in one embodiment, tumorigenic diseases include but are not limited to, mantle cell lymphoma, hereditary & sporadic parathyroid tumors, Medullary thyroid carcinoma, poliverative conditions, colorectal cancer, gliblastoma, Chronic lymphocytic leukaemia, and Breast cancer

[0320] In one embodiment, the disease or disorder is a neurological disease or disorder. For example, in one embodiment, neurological diseases include but are not limited to, Parkinsons diseases, Oculopharyngeal muscular dystrophy, Huntington's disease, Fabry disease, Fragile X syndrome, spinal muscular atrophy, Amyotrophic Lateral Sclerosis, Spinocerebellar ataxia Spinocerebellar ataxia 1, Spinocerebellar ataxia 2, Spinocerebellar ataxia 3, Spinocerebellar ataxia 6, Spinocerebellar ataxia 7, Spinocerebellar ataxia 8, Spinocerebellar ataxia 10, Spinocerebellar ataxia 17, Spinocerebellar ataxia 31, and Alzheimer's disease, .

[0321] In one embodiment, the disease or disorder is a hematological disease or disorder. For example, in one embodiment, hematological diseases include but are not limited to, β-Thalassemia, and α-Thalassemia.

[0322] In one embodiment, the disease or disorder is an infection or immunological disease or disorder. For example, in one embodiment, infection or immunological diseases include but are not limited to, B-cell differentiation, T-cell activation, systemic lupus erythematosus, Wiskott-Aldrich syndrome, Osteoarthris, scleroderma, and IPEX syndrome.

[0323] In one embodiment, the disease or disorder is a musculoskeletal disease or disorder. For example, in one embodiment, infection or immunological diseases include Myotonic dystrophy type 1, Spinal and bulbar muscular atrophy, and Dentatorubral-pallidoluysian atrophy.

[0324] Exemplary diseases or disorders and corresponding targets include, but are not limited to those listed in Table 2. Additional diseases and disorders and corresponding genes are known in the art, for example in Rehfeld et al., Alternations in Polyadenylation and its Implications for Endocrine Disease, Front. Endocrinol. 4:53 (2013), Chang et al., Alternative Polyadenylation in Human Diseases, Endocrinol Metab. 32:413-421 (2017), and Curinha et al., Implications of polyadenylation in health and disease, Nucleus 5:508-519 (2014). Table 2. Diseases or disorders and target geneConditionTargetAdrenal dysfunctionSTARAlzheimer's diseaseABCA3, EIF2B3, MSTO2P, OGDHL, PARP6, SLC33A1, SUPV3L1, TAF3, WASH7P, JAK1, ABCA3, UBR1, ALDOC, C10ord10, GABARAPL2, KAT2A, POLR3A, SH3BGRL2, TIMM23, TMC6, UNC80, WTAPAmyotrophic Lateral SclerosisALS (GGGGCC repeat), CCDC92, CRYAB, MAP7D2, PRPH, RTN4, SEC22B, SNAP25, UCHL1, YWHAB, C14ord2, C6orf203, ALDOC, ARL6IP1, CSDE1, CMC2, GAP43, LDHB, MLLT11, NCL, PFN2, TMOD1, VAMP1B-cell differentiationIGHMB-cell differentiationIGHMBreast cancerCancer, ColorectalDMKN, PDXK, PPIECancer, VariousTP53Chronic lymphocytic lenkaemiaIntronic lociColorectal cancerDMKN, PDXK, PPIEDentatorubral-pallidoluy sian atrophyDRPLA (CAG repeat)Diabetic nephropathyHGRG-14Diabetic nephropathyHRG-14Ectopic Cushing syndromeACTHFabry diseaseα-GalAFragile X SyndromeFMRI, FXTAS (CGG repeat)Friedreich's AtaxiaYSH1GlioblastomaCCND1GlioblastomaCCND1, MECP2Gonadal dysfunctionSTARHuntington's diseaseHTT, HD (CAG repeat)Huntington's disease-like 2HDL2 (CTG repeat)Hypothalamic-pituitary-adrenal axis dysregulationSERTIGF-1 deficiencyIGF-1IPEX syndromeFOP3IPEX syndromeFOXP3IPEX SyndromeFOXP3Mayer-Rokitanski-Küster-HausersyndromeAMHMyotonic Dystrophy Type IDMPK, DM1 (CTG Repeat), DM2 (CCTG repeat)Myotonic Dystrophy Type IIZFN9neonatal diabetesINSNeonatal diabetesINSOculopharyngeal muscular dystrophyCCND1Oculopharyngeal muscular dystrophyPABPN1Parkinson diseaseSNCAParkinson's diseaseSNCAPre-eclampsiaSFLT-1Proliferative conditionsRBX1Proliferative conditionsRBX1Spinal Muscular AtrophySMNSpinocerebellar Ataxia 1ATXN1, SCA1 (CAG repeat)Spinocerebellar Ataxia 10ATXN10, SCA10 (ATTCT repeat)Spinocerebellar Ataxia 17TATA-box binding protein, SCA17 (CAG repeat)Spinocerebellar Ataxia 2ATXN2, SCA2 (CAG repeat)Spinocerebellar Ataxia 3ATXN3, SCA3 (CAG repeat)Spinocerebellar Ataxia 31BEAN1, SCA31 (TGGA repeat)Spinocerebellar Ataxia 6CACNA1A, SCA6 (CAG repeat)Spinocerebellar Ataxia 7TPP1, SCA7 (CAG repeat)Spinocerebellar Ataxia 8ATXN8OS, SCA8 (CTG / CAG repeat)Spinal and bulbar muscular atrophySBMA (CAG repeat)SteroidogenesisSTARSteroidogenesisSTARSystemic lupus erythematosusGIMAP5Systemic lupus erythematosusGIMAP5Systemic lupus erythematosusIRF5T-cell activationNF-ATC1T-cell activationNF-ATC1ThrombophiliaF2Type I diabetesGIMAP5Type I diabetesGIMAP5Type II diabetesTCF7L2Wiskott-Aldrich SyndromeWASα-ThalassaemiaHBA1, HBA2β-ThalassaemiaHBB Amino Acid and Nucleic Acid Sequences

[0325] Table 3 provides a reference of sequences. Table 3SEQ ID NO Type Description 1Amino AcidPspCas13b2Amino AcidPspCas13b Truncation3Amino AcidAspCas13b4Amino AcidAspCas13c5Amino AcidBmaCas13a6Amino AcidBzoCas13b7Amino AcidCamCas13a8Amino AcidCcaCas13b9Amino AcidCga2Cas13a10Amino AcidCgaCas13a11Amino AcidEbaCas13a12Amino AcidEreCas13a13Amino AcidEsCas13d14Amino AcidFbrCas13b15Amino AcidFnbCas13c16Amino AcidFndCas13c17Amino AcidFnfCas13c18Amino AcidFnsCas13c19Amino AcidFpeCas13c20Amino AcidFulCas13c21Amino AcidHheCas13a22Amino AcidLbfCas13a23Amino AcidLbmCas13a24Amino AcidLbnCas13a25Amino AcidLbuCas13a26Amino AcidLseCas13a27Amino AcidLshCas13a28Amino AcidLspCas13a29Amino AcidLwa2cas13a30Amino AcidLwaCas13a31Amino AcidLweCas13a32Amino AcidPauCas13b33Amino AcidPbuCas13b34Amino AcidPgiCas13b35Amino AcidPguCas13b36Amino AcidPin2Cas13b37Amino AcidPin3Cas13b38Amino AcidPinCas13b39Amino AcidPprcas13a40Amino AcidPsaCas13b41Amino AcidPsmCas13b42Amino AcidRanCas13b43Amino AcidRcdCas13a44Amino AcidRcrCas13a45Amino AcidRcsCas13a46Amino AcidUrCas13d47Amino AciddPspCas13b48Amino AciddPspCas13b truncation49Amino AcidCPSF3050Amino AcidWDR3351Amino AcidNUDT2152Amino AcidWorm NudT2153Amino AcidFly NudT2154Amino AcidZebrafish NUDT2155Amino AcidHuman NUDT21 Truncation Mutant56Amino AcidNUDT21 R63S57Amino AcidNUDT21 F103A58Amino AcidNudT21 Tandem Dimer59Amino AcideGFP60Amino AcidmCherry61Amino AcidsfCherry(1-10)62Amino AcidsfCherry(1-10)-L-(11)63Amino Acid7xS1164Amino AcidsfGFP65Amino AcidsfGFP(1-10)66Amino AcidsfGFP(1-10)-L-(11)67Amino AcidLinker sequence 168Amino AcidLinker sequence 269Amino AcidLinker sequence 370Amino AcidLinker sequence 471Amino AcidLinker sequence 572Amino AcidLinker sequence 673Amino AcidLinker sequence 774Amino Acid3xFlag75Amino AcidTy1 NLS76Amino AcidTy2 NLS77Amino AcidMAK1178Amino Acid1xSV4079Amino Acid3xSV4080Amino AcidNPM81Amino AcidSTH182Amino AcidINO483Amino AcidTy1-like NLS O28090-084Amino AcidTy1-like NLS O50087-085Amino AcidTy1-like NLS O58353-086Amino AcidTy1-like NLS Q57602-087Amino AcidTy1-like NLS Q6L1X9-088Amino AcidTy1-like NLS A0K3M1-089Amino AcidTy1-like NLS A0LYZ1-090Amino AcidTy1-like NLS A1B022-091Amino AcidTy1-like NLS A1V8A7-092Amino AcidTy1-like NLS A1 VIP6-093Amino AcidTy1-like NLS A2RDW6-094Amino AcidTy1-like NLS A2S7H2-095Amino AcidTy1-like NLS A3MRV0-096Amino AcidTy1-like NLS A3NEI3-097Amino AcidTy1-like NLS A3P0B7-098Amino AcidTy1-like NLS A4JAN6-099Amino AcidTy1-like NLS A4SUV7-0100Amino AcidTy1-like NLS A5FP03-0101Amino AcidTy1-like NLS A5ILZ2-0102Amino AcidTy1-like NLS A6GY20-0103Amino AcidTy1-like NLS A6LLI5-0104Amino AcidTy1-like NLS A6LQX4-0105Amino AcidTy1-like NLS A8F6X2-0106Amino AcidTy1-like NLS A8G6B7-0107Amino AcidTy1-like NLS A9ADI9-0108Amino AcidTy1-like NLS A91J08-0109Amino AcidTy1-like NLS A9IXA1-0110Amino AcidTy1-like NLS A9NEN2-0111Amino AcidTy1-like NLS B0S140-0112Amino AcidTy1-like NLS B1JU18-0113Amino AcidTy1-like NLS B 1LBA1-0114Amino AcidTy1-like NLS B1 W354-0115Amino AcidTy1-like NLS B1XSP7-0116Amino AcidTy1-like NLS B1YRC6-0117Amino AcidTy1-like NLS B2JIH0-0118Amino AcidTy1-like NLS B2T755-0119Amino AcidTy1-like NLS B2UEM3-0120Amino AcidTy1-like NLS B3PLU0-0121Amino AcidTy1-like NLS B3R7T2-0122Amino AcidTy1-like NLS B4E5B6-0123Amino AcidTy1-like NLS B4S3C9-0124Amino AcidTy1-like NLS B7IHT4-0125Amino AcidTy1-like NLS B8E0X6-0126Amino AcidTy1-like NLS B9K7WO-0127Amino AcidTy1-like NLS C1A494-0128Amino AcidTy1-like NLS C5CE41-0129Amino AcidTy1-like NLS O88058-0130Amino AcidTy1-like NLS P0DG92-0131Amino AcidTy1-like NLS P0DG93-0132Amino AcidTy1-like NLS P60554-0133Amino AcidTy1-like NLS P67354-0134Amino AcidTy1-like NLS P75311-0135Amino AcidTy1-like NLS P75471-0136Amino AcidTy1-like NLS P94372-0137Amino AcidTy1-like NLS Q056Y0-0138Amino AcidTy1-like NLS Q057D7-0139Amino AcidTy1-like NLS Q0AYB7-0140Amino AcidTy1-like NLS Q0BJ50-0141Amino AcidTy1-like NLS Q0K610-0142Amino AcidTy1-like NLS Q0STA4-0143Amino AcidTy1-like NLS Q0STL9-0144Amino AcidTy1-like NLS Q0TQV7-0145Amino AcidTy1-like NLS Q0TR88-0146Amino AcidTy1-like NLS Q12GX5-0147Amino AcidTy1-like NLS Q13TG6-0148Amino AcidTy1-like NLS Q1AWG1-0149Amino AcidTy1-like NLS Q1BRU4-0150Amino AcidTy1-like NLS Q1J5X5-0151Amino AcidTy1-like NLS Q1JAY8-0152Amino AcidTy1-like NLS Q1JG57-0153Amino AcidTy1-like NLS Q1JL34-0154Amino AcidTy1-like NLS Q1LI28-0155Amino AcidTy1-like NLS Q2L2H3-0156Amino AcidTy1-like NLS Q2NIH1-0157Amino AcidTy1-like NLS Q2SU23-0158Amino AcidTy1-like NLS Q39KH1-0159Amino AcidTy1-like NLS Q3JMQ8-0160Amino AcidTy1-like NLS Q3YRL8-0161Amino AcidTy1-like NLS Q46WD9-0162Amino AcidTy1-like NLS Q48SQ4-0163Amino AcidTy1-like NLS Q49418-0164Amino AcidTy1-like NLS Q56307-0165Amino AcidTy1-like NLS Q5LEQ4-0166Amino AcidTy1-like NLS Q5WEJ7-0167Amino AcidTy1-like NLS Q5XBA0-0168Amino AcidTy1-like NLS Q62GK1-0169Amino AcidTy1-like NLS Q63Q07-0170Amino AcidTy1-like NLS Q64VP0-0171Amino AcidTy1-like NLS Q6G3V1-0172Amino AcidTy1-like NLS Q6G5M0-0173Amino AcidTy1-like NLS Q6LLQ8-0174Amino AcidTy1-like NLS Q6MDC1-0175Amino AcidTy1-like NLS Q6MDH4-0176Amino AcidTy1-like NLS Q6ME08-0177Amino AcidTy1-like NLS Q73PH4-0178Amino AcidTy1-like NLS Q7MAD1-0179Amino AcidTy1-like NLS Q7UP72-0180Amino AcidTy1-like NLS Q7VTD6-0181Amino AcidTy1-like NLS Q7W2F9-0182Amino AcidTy1-like NLS Q7WRC8-0183Amino AcidTy1-like NLS Q828D0-0184Amino AcidTy1-like NLS Q895M9-0185Amino AcidTy1-like NLS Q8AAP0-0186Amino AcidTy1-like NLS Q8D1X2-0187Amino AcidTy1-like NLS Q8K908-0188Amino AcidTy1-like NLS Q8P0C9-0189Amino AcidTy1-like NLS Q8XKR1-0190Amino AcidTy1-like NLS Q8XL46-0191Amino AcidTy1-like NLS Q8XV09-0192Amino AcidTy1-like NLS Q93Q47-0193Amino AcidTy1-like NLS Q9L0Q6-0194Amino AcidTy1-like NLS Q9L0Q6-1195Amino AcidTy1-like NLS Q9L0Q6-2196Amino AcidTy1-like NLS Q9L0Q6-3197Amino AcidTy1-like NLS Q9L0Q6-4198Amino AcidTy1-like NLS Q9L0Q6-5199Amino AcidTy1-like NLS Q9L0Q6-6200Amino AcidTy1-like NLS Q9X1S8-0201Amino AcidTy1-like NLS A1CNV8-0202Amino AcidTy1-like NLS A1D1R8-0203Amino AcidTy1-like NLS A1D731-0204Amino AcidTy1-like NLS A2QAX7-0205Amino AcidTy1-like NLS A3LQ55-0206Amino AcidTy1-like NLS A5DGY0-0207Amino AcidTy1-like NLS A5DKW3-0208Amino AcidTy1-like NLS A5DLG8-0209Amino AcidTy1-like NLS A5DY34-0210Amino AcidTy1-like NLS A6RBB0-0211Amino AcidTy1-like NLS A6RMZ2-0212Amino AcidTy1-like NLS A6ZL85-0213Amino AcidTy1-like NLS A6ZZJ1-0214Amino AcidTy1-like NLS A7E4K0-0215Amino AcidTy1-like NLS G0S8I1-0216Amino AcidTy1-like NLS 013527-0217Amino AcidTy1-like NLS 013535-0218Amino AcidTy1-like NLS 013658-0219Amino AcidTy1-like NLS 014064-0220Amino AcidTy1-like NLS 014076-0221Amino AcidTy1-like NLS O42668-0222Amino AcidTy1-like NLS 043068-0223Amino AcidTy1-like NLS O74777-0224Amino AcidTy1-like NLS O74862-0225Amino AcidTy1-like NLS O94383-0226Amino AcidTy1-like NLS O94487-0227Amino AcidTy1-like NLS O94585-0228Amino AcidTy1-like NLS O94652-0229Amino AcidTy1-like NLS P0C2I2-0230Amino AcidTy1-like NLS P0C2I3-0231Amino AcidTy1-like NLS P0C2I5-0232Amino AcidTy1-like NLS P0C2I6-0233Amino AcidTy1-like NLS P0C2I7-0234Amino AcidTy1-like NLS P0C2I9-0235Amino AcidTy1-like NLS P0C2J0-0236Amino AcidTy1-like NLS P0C2J1-0237Amino AcidTy1-like NLS P0C2J3-0238Amino AcidTy1-like NLS P0C2J5-0239Amino AcidTy1-like NLS P0CM98-0240Amino AcidTy1-like NLS P0CM99-0241Amino AcidTy1-like NLS P0CX63-0242Amino AcidTy1-like NLS P0CX64-0243Amino AcidTy1-like NLS P13902-0244Amino AcidTy1-like NLS P14746-0245Amino AcidTy1-like NLS P20484-0246Amino AcidTy1-like NLS P22936-0247Amino AcidTy1-like NLS P25384-0248Amino AcidTy1-like NLS P32597-0249Amino AcidTy1-like NLS P36006-0250Amino AcidTy1-like NLS P36080-0251Amino AcidTy1-like NLS P38112-0252Amino AcidTy1-like NLS P47098-0253Amino AcidTy1-like NLS P47100-0254Amino AcidTy1-like NLS P51599-0255Amino AcidTy1-like NLS P53119-0256Amino AcidTy1-like NLS P53123-0257Amino AcidTy1-like NLS P53125-0258Amino AcidTy1-like NLS Q01301-0259Amino AcidTy1-like NLS Q03434-0260Amino AcidTy1-like NLS Q03494-0261Amino AcidTy1-like NLS Q03612-0262Amino AcidTy1-like NLS Q03619-0263Amino AcidTy1-like NLS Q03707-0264Amino AcidTy1-like NLS Q03855-0265Amino AcidTy1-like NLS Q04214-0266Amino AcidTy1-like NLS Q04500-0267Amino AcidTy1-like NLS Q04670-0268Amino AcidTy1-like NLS Q04711-0269Amino AcidTy1-like NLS Q06132-0270Amino AcidTy1-like NLS Q07163-0271Amino AcidTy1-like NLS Q07509-0272Amino AcidTy1-like NLS Q07791-0273Amino AcidTy1-like NLS Q07793-0274Amino AcidTy1-like NLS Q09094-0275Amino AcidTy1-like NLS Q09180-0276Amino AcidTy1-like NLS Q09180-1277Amino AcidTy1-like NLS Q09180-2278Amino AcidTy1-like NLS Q09863-0279Amino AcidTy1-like NLS Q0U8V9-0280Amino AcidTy1-like NLS Q12088-0281Amino AcidTy1-like NLS Q12112-0282Amino AcidTy1-like NLS Q12113-0283Amino AcidTy1-like NLS Q12141-0284Amino AcidTy1-like NLS Q12193-0285Amino AcidTy1-like NLS Q12269-0286Amino AcidTy1-like NLS Q12273-0287Amino AcidTy1-like NLS Q12316-0288Amino AcidTy1-like NLS Q12337-0289Amino AcidTy1-like NLS Q12339-0290Amino AcidTy1-like NLS Q12414-0291Amino AcidTy1-like NLS Q12472-0292Amino AcidTy1-like NLS Q12490-0293Amino AcidTy1-like NLS Q12491-0294Amino AcidTy1-like NLS Q12501-0295Amino AcidTy1-like NLS Q1DNW5-0296Amino AcidTy1-like NLS Q1EA54-0297Amino AcidTy1-like NLS Q2HFA6-0298Amino AcidTy1-like NLS Q2HFA6-1299Amino AcidTy1-like NLS Q2UQI6-0300Amino AcidTy1-like NLS Q4HZ42-0301Amino AcidTy1-like NLS Q4P613-0302Amino AcidTy1-like NLS Q4WHF8-0303Amino AcidTy1-like NLS Q4WRV2-0304Amino AcidTy1-like NLS Q4WXQ7-0305Amino AcidTy1-like NLS Q5A2K0-0306Amino AcidTy1-like NLS Q5A310-0307Amino AcidTy1-like NLS Q5ACW8-0308Amino AcidTy1-like NLS Q5B6K3-0309Amino AcidTy1-like NLS Q6BXL7-0310Amino AcidTy1-like NLS Q6C1L3-0311Amino AcidTy1-like NLS Q6C233-0312Amino AcidTy1-like NLS Q6C2J1-0313Amino AcidTy1-like NLS Q6C7C0-0314Amino AcidTy1-like NLS Q6CJY0-0315Amino AcidTy1-like NLS Q6CJY0-1316Amino AcidTy1-like NLS Q6FML5-0317Amino AcidTy1-like NLS Q75F02-0318Amino AcidTy1-like NLS Q7S2A9-0319Amino AcidTy1-like NLS Q7S9J4-0320Amino AcidTy1-like NLS Q7SFJ3-0321Amino AcidTy1-like NLS Q875K1-0322Amino AcidTy1-like NLS Q8SUT1-0323Amino AcidTy1-like NLS Q8SVI7-0324Amino AcidTy1-like NLS Q8SVI7-1325Amino AcidTy1-like NLS Q92393-0326Amino AcidTy1-like NLS Q99109-0327Amino AcidTy1-like NLS Q99231-0328Amino AcidTy1-like NLS Q99337-0329Amino AcidTy1-like NLS Q9USK2-0330Amino AcidTy1-like NLS Q9UTQ5-0331Amino AcidTy1-like NLS A7MD48-0332Amino AcidTy1-like NLS 015446-0333Amino AcidTy1-like NLS 015446-1334Amino AcidTy1-like NLS 015446-2335Amino AcidTy1-like NLS 043148-0336Amino AcidTy1-like NLS 060271-0337Amino AcidTy1-like NLS 075128-0338Amino AcidTy1-like NLS O75400-0339Amino AcidTy1-like NLS 075691-0340Amino AcidTy1-like NLS O75937-0341Amino AcidTy1-like NLS 076021-0342Amino AcidTy1-like NLS O94964-0343Amino AcidTy1-like NLS P23497-0344Amino AcidTy1-like NLS P30414-0345Amino AcidTy1-like NLS P42081-0346Amino AcidTy1-like NLS P46100-0347Amino AcidTy1-like NLS P51608-0348Amino AcidTy1-like NLS P59797-0349Amino AcidTy1-like NLS P82979-0350Amino AcidTy1-like NLS Q12830-0351Amino AcidTy1-like NLS Q13409-0352Amino AcidTy1-like NLS Q13427-0353Amino AcidTy1-like NLS Q15361-0354Amino AcidTy1-like NLS Q15361-1355Amino AcidTy1-like NLS Q53SF7-0356Amino AcidTy 1-like NLS Q5M9Q1-0357Amino AcidTy 1-like NLS Q5T310-0358Amino AcidTy 1-like NLS Q5T3I0-1359Amino AcidTy 1-like NLS Q68D10-0360Amino AcidTy 1-like NLS Q6IPR3-0361Amino AcidTy 1-like NLS Q6PD62-0362Amino AcidTy1-like NLS Q6PD62-1363Amino AcidTy 1-like NLS Q6PD62-2364Amino AcidTy1-like NLS Q6S8J7-0365Amino AcidTy1-like NLS Q6ZU65-0366Amino AcidTy1-like NLS Q7Z7B0-0367Amino AcidTy 1-like NLS Q8N9E0-0368Amino AcidTy 1-like NLS Q8NCU4-0369Amino AcidTy1-like NLS Q8NFU7-0370Amino AcidTy 1-like NLS Q96DY2-0371Amino AcidTy1-like NLS Q96GD3-0372Amino AcidTy 1-like NLS Q96P65-0373Amino AcidTy 1-like NLS Q96QC0-0374Amino AcidTy 1-like NLS Q9BQG0-0375Amino AcidTy 1-like NLS Q9BQG0-1376Amino AcidTy 1-like NLS Q9BRU9-0377Amino AcidTy 1-like NLS Q9H0S4-0378Amino AcidTy 1-like NLS Q9H6F5-0379Amino AcidTy1-like NLS Q9HCK1-0380Amino AcidTy1-like NLS Q9HCK8-0381Amino AcidTy1-like NLS Q9NPI1-0382Amino AcidTy 1-like NLS Q9NSV4-0383Amino AcidTy 1-like NLS Q9NUL3-0384Amino AcidTy1-like NLS Q9NWT1-0385Amino AcidTy 1-like NLS Q9NX58-0386Amino AcidTy1-like NLS Q9UGU5-0387Amino AcidTy1-like NLS Q9UNS1-0388Amino AcidTy 1-like NLS Q9Y2X3-0389Amino AcidTy 1-like NLS Q9Y6X0-0390Amino AcidTy1-like NLS A0A1I8M2I8-0391Amino AcidTy1-like NLS A1XDC0-0392Amino AcidTy1-like NLS A7S6A5-0393Amino AcidTy 1-like NLS A8XI07-0394Amino AcidTy1-like NLS A8XI07-1395Amino AcidTy 1-like NLS C0HKU9-0396Amino AcidTy 1-like NLS C6KTD2-0397Amino AcidTy 1-like NLS 016140-0398Amino AcidTy1-like NLS 017828-0399Amino AcidTy1-like NLS 017966-0400Amino AcidTy 1-like NLS 044410-0401Amino AcidTy1-like NLS 044410-1402Amino AcidTy 1-like NLS O45244-0403Amino AcidTy 1-like NLS P0DP78-0404Amino AcidTy 1-like NLS P0DP78-1405Amino AcidTy1-like NLS P0DP79-0406Amino AcidTy1-like NLS P0DP79-1407Amino AcidTy 1-like NLS P0DP80-0408Amino AcidTy 1-like NLS P0DP80-1409Amino AcidTy 1-like NLS P0DP81-0410Amino AcidTy 1-like NLS P0DP81-1411Amino AcidTy 1-like NLS P14196-0412Amino AcidTy 1-like NLS P22058-0413Amino AcidTy 1-like NLS P26023-0414Amino AcidTy1-like NLS P26991-0415Amino AcidTy1-like NLS P35978-0416Amino AcidTy 1-like NLS P46758-0417Amino AcidTy1-like NLS P46758-1418Amino AcidTy 1-like NLS P46867-0419Amino AcidTy 1-like NLS P54644-0420Amino AcidTy1-like NLS P54812-0421Amino AcidTy1-like NLS P83212-0422Amino AcidTy1-like NLS Q04621-0423Amino AcidTy 1-like NLS Q08696-0424Amino AcidTy 1-like NLS Q08696-1425Amino AcidTy 1-like NLS Q08696-2426Amino AcidTy 1-like NLS Q08696-3427Amino AcidTy 1-like NLS Q08696-4428Amino AcidTy 1-like NLS Q08696-5429Amino AcidTy 1-like NLS Q08696-6430Amino AcidTy 1-like NLS Q09223-0431Amino AcidTy 1-like NLS Q09595-0432Amino AcidTy1-like NLS Q1ELU8-0433Amino AcidTy1-like NLS Q23120-0434Amino AcidTy1-like NLS Q23272-0435Amino AcidTy1-like NLS Q24537-0436Amino AcidTy1-like NLS Q27450-0437Amino AcidTy1-like NLS Q29DY1-0438Amino AcidTy 1-like NLS Q4N4T9-0439Amino AcidTy 1-like NLS Q54QQ2-0440Amino AcidTy1-like NLS Q54QQ2-1441Amino AcidTy1-like NLS Q54S20-0442Amino AcidTy 1-like NLS Q54US6-0443Amino AcidTy1-like NLS Q54VU4-0444Amino AcidTy 1-like NLS Q54XP6-0445Amino AcidTy1-like NLS Q551H0-0446Amino AcidTy1-like NLS Q557G1-0447Amino AcidTy 1-like NLS Q55CE0-0448Amino AcidTy 1-like NLS Q61R02-0449Amino AcidTy 1-like NLS Q75JP5-0450Amino AcidTy1-like NLS Q8I5P7-0451Amino AcidTy1-like NLS Q815P7-1452Amino AcidTy1-like NLS Q8IBP1-0453Amino AcidTy1-like NLS Q8ILR9-0454Amino AcidTy1-like NLS Q93591-0455Amino AcidTy 1-like NLS Q95Y36-0456Amino AcidTy 1-like NLS Q9NBL2-0457Amino AcidTy 1-like NLS Q9NDE8-0458Amino AcidTy1-like NLS Q9NDE8-1459Amino AcidTy 1-like NLS Q9NDE8-2460Amino AcidTy 1-like NLS Q9V5P6-0461Amino AcidTy 1-like NLS Q9VDS6-0462Amino AcidTy1-like NLS Q9VGW1-0463Amino AcidTy 1-like NLS Q9VH89-0464Amino AcidTy 1-like NLS Q9VKM6-0465Amino AcidTy1-like NLS Q9VNH1-0466Amino AcidTy1-like NLS Q9W261-0467Amino AcidTy1-like NLS E1B7L7-0468Amino AcidTy1-like NLS Q08DU1-0469Amino AcidTy 1-like NLS Q0III3-0470Amino AcidTy 1-like NLS Q 17QH9-0471Amino AcidTy1-like NLS Q29S22-0472Amino AcidTy1-like NLS Q2KIQ2-0473Amino AcidTy1-like NLS Q2KJE1-0474Amino AcidTy1-like NLS Q2KJE1-1475Amino AcidTy1-like NLS Q2TBX7-0476Amino AcidTy1-like NLS Q4R7K1-0477Amino AcidTy1-like NLS Q4R8Y5-0478Amino AcidTy1-like NLS Q58DE2-0479Amino AcidTy1-like NLS Q58DU0-0480Amino AcidTy1-like NLS Q5E9U4-0481Amino AcidTy 1-like NLS Q5NVM2-0482Amino AcidTy 1-like NLS Q5R4V4-0483Amino AcidTy 1-like NLS Q5R8B0-0484Amino AcidTy 1-like NLS Q5RB69-0485Amino AcidTy 1-like NLS Q5RCE6-0486Amino AcidTy1-like NLS Q5TM61-0487Amino AcidTy1-like NLS Q767K9-0488Amino AcidTy 1-like NLS Q7YQM3-0489Amino AcidTy 1-like NLS Q7YQM4-0490Amino AcidTy1-like NLS Q7YR38-0491Amino AcidTy1-like NLS Q95KD7-0492Amino AcidTy1-like NLS Q95LG8-0493Amino AcidTy1-like NLS Q9N1Q7-0494Amino AcidTy1-like NLS A2WSD3-0495Amino AcidTy1-like NLS A2XVF7-0496Amino AcidTy1-like NLS A2XVF7-1497Amino AcidTy1-like NLS A2XVF7-2498Amino AcidTy1-like NLS A2XVF7-3499Amino AcidTy1-like NLS A3AVH5-0500Amino AcidTy1-like NLS A3AVH5-1501Amino AcidTy1-like NLS A3AVH5-2502Amino AcidTy1-like NLS A3AVH5-3503Amino AcidTy 1-like NLS A4QJZ0-0504Amino AcidTy 1-like NLS A4QK78-0505Amino AcidTy1-like NLS A4QKG5-0506Amino AcidTy 1-like NLS A4QKQ3-0507Amino AcidTy 1-like NLS A6MN03-0508Amino AcidTy 1-like NLS A8MS85-0509Amino AcidTy 1-like NLS A9XMT3-0510Amino AcidTy 1-like NLS B8YIE8-0511Amino AcidTy 1-like NLS F4HVZ5-0512Amino AcidTy 1-like NLS F4IQK5-0513Amino AcidTy 1-like NLS F4IQK5-1514Amino AcidTy1-like NLS 022812-0515Amino AcidTy 1-like NLS 049323-0516Amino AcidTy1-like NLS 064571-0517Amino AcidTy 1-like NLS O64639-0518Amino AcidTy1-like NLS 064639-1519Amino AcidTy 1-like NLS O64639-2520Amino AcidTy 1-like NLS 065743-0521Amino AcidTy1-like NLS 081072-0522Amino AcidTy 1-like NLS P09975-0523Amino AcidTy 1-like NLS P0C262-0524Amino AcidTy 1-like NLS P29345-0525Amino AcidTy 1-like NLS P50888-0526Amino AcidTy 1-like NLS P51269-0527Amino AcidTy1-like NLS P51430-0528Amino AcidTy 1-like NLS Q06FP6-0529Amino AcidTy1-like NLS Q06FP6-1530Amino AcidTy1-like NLS Q06FP6-2531Amino AcidTy 1-like NLS Q06R72-0532Amino AcidTy 1-like NLS Q06R98-0533Amino AcidTy1-like NLS Q1KVQ9-0534Amino AcidTy1-like NLS Q1XDL7-0535Amino AcidTy1-like NLS Q38873-0536Amino AcidTy 1-like NLS Q3E8X3-0537Amino AcidTy1-like NLS Q3ZJ77-0538Amino AcidTy1-like NLS Q42438-0539Amino AcidTy1-like NLS Q4V3E0-0540Amino AcidTy1-like NLS Q66GN2-0541Amino AcidTy1-like NLS Q6K5K2-0542Amino AcidTy1-like NLS Q6YS30-0543Amino AcidTy 1-like NLS Q84WK0-0544Amino AcidTy1-like NLS Q84Y18-0545Amino AcidTy1-like NLS Q8H991-0546Amino AcidTy 1-like NLS Q8RWY7-0547Amino AcidTy1-like NLS Q8RWY7-1548Amino AcidTy1-like NLS Q8VZ67-0549Amino AcidTy1-like NLS Q8VZN4-0550Amino AcidTy 1-like NLS Q8W0K2-0551Amino AcidTy 1-like NLS Q8W490-0552Amino AcidTy 1-like NLS Q9CAE4-0553Amino AcidTy 1-like NLS Q9FMZ4-0554Amino AcidTy 1-like NLS Q9FMZ4-1555Amino AcidTy 1-like NLS Q9FRI0-0556Amino AcidTy 1-like NLS Q9LKI5-0557Amino AcidTy1-like NLS Q9LUJ5-0558Amino AcidTy1-like NLS Q9LUR0-0559Amino AcidTy 1-like NLS Q9LVU8-0560Amino AcidTy 1-like NLS Q9LVU8-1561Amino AcidTy 1-like NLS Q9LYK7-0562Amino AcidTy 1-like NLS Q9M020-0563Amino AcidTy1-like NLS Q9M1L7-0564Amino AcidTy 1-like NLS Q9M3V8-0565Amino AcidTy1-like NLS Q9SRQ3-0566Amino AcidTy 1-like NLS Q9ZPV5-0567Amino AcidTy1-like NLS B1AQJ2-0568Amino AcidTy 1-like NLS D3ZUI5-0569Amino AcidTy 1-like NLS D4A666-0570Amino AcidTy1-like NLS E1U8D0-0571Amino AcidTy 1-like NLS G3V8T1-0572Amino AcidTy1-like NLS 035821-0573Amino AcidTy 1-like NLS O88487-0574Amino AcidTy 1-like NLS O88665-0575Amino AcidTy1-like NLS P61364-0576Amino AcidTy 1-like NLS P61365-0577Amino AcidTy 1-like NLS P83858-0578Amino AcidTy1-like NLS P83861-0579Amino AcidTy 1-like NLS Q00566-0580Amino AcidTy1-like NLS Q05CL8-0581Amino AcidTy 1-like NLS Q09XV5-0582Amino AcidTy1-like NLS Q3TFK5-0583Amino AcidTy1-like NLS Q3TFK5-1584Amino AcidTy1-like NLS Q3TFK5-2585Amino AcidTy1-like NLS Q3TYA6-0586Amino AcidTy 1-like NLS Q3UMF0-0587Amino AcidTy 1-like NLS Q498U4-0588Amino AcidTy 1-like NLS Q4V7C4-0589Amino AcidTy 1-like NLS Q4V8G7-0590Amino AcidTy 1-like NLS Q50515-0591Amino AcidTy 1-like NLS Q562C7-0592Amino AcidTy1-like NLS Q566R3-0593Amino AcidTy 1-like NLS Q566R3-1594Amino AcidTy 1-like NLS Q566R3-2595Amino AcidTy 1-like NLS Q58A65-0596Amino AcidTy1-like NLS Q5NBX1-0597Amino AcidTy 1-like NLS Q5XG71-0598Amino AcidTy 1-like NLS Q5XI01-0599Amino AcidTy 1-like NLS Q5XIB5-0600Amino AcidTy 1-like NLS Q5XIR6-0601Amino AcidTy 1-like NLS Q60848-0602Amino AcidTy1-like NLS Q62018-0603Amino AcidTy1-like NLS Q62018-1604Amino AcidTy1-like NLS Q62187-0605Amino AcidTy1-like NLS Q62871-0606Amino AcidTy 1-like NLS Q63520-0607Amino AcidTy 1-like NLS Q642C0-0608Amino AcidTy 1-like NLS Q68SB1-0609Amino AcidTy1-like NLS Q6AYK5-0610Amino AcidTy 1-like NLS Q6NZB0-0611Amino AcidTy1-like NLS Q76KJ5-0612Amino AcidTy1-like NLS Q76KJ5-1613Amino AcidTy1-like NLS Q76KJ5-2614Amino AcidTy1-like NLS Q78WZ7-0615Amino AcidTy1-like NLS Q78WZ7-1616Amino AcidTy1-like NLS Q7TNB4-0617Amino AcidTy1-like NLS Q7TPV4-0618Amino AcidTy1-like NLS Q80WC1-0619Amino AcidTy1-like NLS Q80Z37-0620Amino AcidTy1-like NLS Q811R2-0621Amino AcidTy1-like NLS Q8BKA3-0622Amino AcidTy 1-like NLS Q8CJ67-0623Amino AcidTy1-like NLS Q8K214-0624Amino AcidTy1-like NLS Q8K4T4-0625Amino AcidTy 1-like NLS Q8R5F3-0626Amino AcidTy 1-like NLS Q91X13-0627Amino AcidTy1-like NLS Q9CS72-0628Amino AcidTy 1-like NLS Q9CVI2-0629Amino AcidTy 1-like NLS Q9CWX9-0630Amino AcidTy 1-like NLS Q9CZX5-0631Amino AcidTy1-like NLS Q9D1J3-0632Amino AcidTy1-like NLS Q9D3V1-0633Amino AcidTy 1-like NLS Q9DBQ9-0634Amino AcidTy 1-like NLS Q9JIX5-0635Amino AcidTy 1-like NLS Q9JJ80-0636Amino AcidTy 1-like NLS Q9JJ89-0637Amino AcidTy1-like NLS Q9R1C7-0638Amino AcidTy 1-like NLS Q9R1X4-0639Amino AcidTy1-like NLS Q9Z180-0640Amino AcidTy1-like NLS Q9Z207-0641Amino AcidTy 1-like NLS Q9Z2D6-0642Amino AcidTy1-like NLS A0A1L8GSA2-0643Amino AcidTy 1-like NLS A0JP82-0644Amino AcidTy1-like NLS A1A5I1-0645Amino AcidTy 1-like NLS A1L2T6-0646Amino AcidTy1-like NLS A2RUV0-0647Amino AcidTy1-like NLS A9JRD8-0648Amino AcidTy 1-like NLS E7F568-0649Amino AcidTy 1-like NLS F1QFU0-0650Amino AcidTy1-like NLS F1QWK4-0651Amino AcidTy 1-like NLS K9JHZ4-0652Amino AcidTy 1-like NLS P07193-0653Amino AcidTy1-like NLS P0CB65-0654Amino AcidTy1-like NLS P12957-0655Amino AcidTy1-like NLS P13505-0656Amino AcidTy1-like NLS P21783-0657Amino AcidTy1-like NLS Q28BS0-0658Amino AcidTy1-like NLS Q28BS0-1659Amino AcidTy 1-like NLS Q28G05-0660Amino AcidTy 1-like NLS Q32N87-0661Amino AcidTy 1-like NLS Q3KPW4-0662Amino AcidTy 1-like NLS Q4QR29-0663Amino AcidTy 1-like NLS Q4QR29-1664Amino AcidTy 1-like NLS Q5BL56-0665Amino AcidTy 1-like NLS Q5XJK9-0666Amino AcidTy1-like NLS Q5ZIJ0-0667Amino AcidTy 1-like NLS Q640I9-0668Amino AcidTy 1-like NLS Q6DEU9-0669Amino AcidTy 1-like NLS Q6DEU9-1670Amino AcidTy1-like NLS Q6DEU9-2671Amino AcidTy 1-like NLS Q6DK85-0672Amino AcidTy 1-like NLS Q6DRI7-0673Amino AcidTy1-like NLS Q6DRL5-0674Amino AcidTy1-like NLS Q6NV26-0675Amino AcidTy1-like NLS Q6NWI1-0676Amino AcidTy 1-like NLS Q6NYJ3-0677Amino AcidTy 1-like NLS Q6P4K1-0678Amino AcidTy1-like NLS Q6WKW9-0679Amino AcidTy 1-like NLS Q7ZUF2-0680Amino AcidTy1-like NLS Q7ZW47-0681Amino AcidTy 1-like NLS Q7ZXZ0-0682Amino AcidTy 1-like NLS Q7ZXZ0-1683Amino AcidTy 1-like NLS Q7ZYR8-0684Amino AcidTy 1-like NLS Q8AVQ6-0685Amino AcidTy1-like NLS Q9DE07-0686Amino AcidTy 1-like NLS P03086-0687Amino AcidTy 1-like NLS P09814-0688Amino AcidTy1-like NLS P0CK10-0689Amino AcidTy1-like NLS P15075-0690Amino AcidTy1-like NLS P51724-0691Amino AcidTy1-like NLS P52344-0692Amino AcidTy1-like NLS P52531-0693Amino AcidTy1-like NLS Q5UP41-0694Amino AcidTy1-like NLS Q9DUC0-0695Amino AcidTy 1-like NLS Q9XJS3-0696Amino AciddPspCas13b-CPSF30 fusion697Amino AciddPspCas13b-WDR33 fusion698Amino AciddPspCas13b- NUDT21 fusion699Amino AcidHlightR Green700Amino AcidHlightR Red701Amino AcidHiLightR-S11702Amino AcidEraseR703Amino AcidMBNL1704Amino AcidmCherry-MBNL1705Nucleic AcidPspCas13b706Nucleic AcidPspCas13b Truncation707Nucleic AciddPspCas13b708Nucleic AciddPspCas13b truncation709Nucleic AcidCPSF30710Nucleic AcidWDR33711Nucleic AcidNUDT21712Nucleic AcidWorm NudT21713Nucleic AcidFly NudT21714Nucleic AcidHuman NUDT21 Truncation Mutant715Nucleic AcidNUDT21 R63S716Nucleic AcidNUDT21 F103A717Nucleic AcidNudT21 Tandem Dimer718Nucleic AcideGFP719Nucleic AcidmCherry720Nucleic Acid7xS11721Nucleic AcidsfGFP722Nucleic AcidLinker sequence 1723Nucleic Acid3xFlag724Nucleic AcidTy1 NLS725Nucleic AciddPspCas13b-CPSF30 fusion726Nucleic AciddPspCas13b-WDR33 fusion727Nucleic AciddPspCas13b- NUDT21 fusion728Nucleic AcidHlightR Green729Nucleic AcidHlightR Red730Nucleic AcidHiLightR-S11731Nucleic AcidEraseR732Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter733Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter734Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter735Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter736Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter737Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter738Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter739Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter740Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter741Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter742Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter743Nucleic Acid3' RACE clone isolated from Postscriptr-targeted sfGFPapa reporter744Nucleic AcidsfGFPapa intronic crRNA target sequence745Nucleic AcidHuman SREBP1 intronic PAS crRNA target sequence746Nucleic Acid3' RACE Oligo d(T)747Nucleic Acid3' RACE Oligo dT Nested Primer 1748Nucleic Acid3' RACE Oligo dT Nested Primer 2749Nucleic Acid3' RACE SREBP1 Gene Specific Primer 1750Nucleic Acid3' RACE SREBP1 Gene Specific Primer 2751Nucleic Acid3' RACE sfGFPapa Gene Specific Primer 1752Nucleic Acid3' RACE sfGFPapa Gene Specific Primer 2753Nucleic AcidSREBP1 upstream qRT-PCT F Primer754Nucleic AcidSREBP1 upstream qRT-PCT R Primer755Nucleic AcidSREBP1 downstream qRT-PCT F Primer756Nucleic AcidSREBP1 downstream qRT-PCT R Primer757Nucleic AcidLDLR qRT-PCT F Primer758Nucleic AcidLDLR qRT-PCT R Primer759Nucleic AcidsfGFPapa reporter760Nucleic AcidMBNL1761Nucleic AcidmCherry-MBNL1762Nucleic AcidPspCas13b crRNA target sequence CASx9763Nucleic AcidPspCas13b crRNA target sequence CASx9-f2764Nucleic AcidPspCas13b crRNA target sequence CASx9-f3765Nucleic AcidPrimer PspCas13b_A133H766Nucleic AcidPrimer PspCas13b_A1058H767Nucleic AcidsfCherryapa Reporter768Nucleic AcidsfGFP crRNA target sequence vector769Nucleic AcidsfGFP crRNA target sequence -4770Nucleic AcidsfGFP crRNA target sequence -6771Nucleic AcidsfGFP crRNA target sequence +6772Nucleic AcidsfGFP crRNA target sequence +9773Nucleic AcidsfGFP crRNA target sequence +16774Nucleic AcidSREBP1775Nucleic AcidSREBP1Δ776Nucleic AcidPostscriptr-induced polyadenylated transcript777Nucleic AcidSREBP1C-PS778Nucleic AcidPspCas13b crRNA direct repeat sequence779Nucleic AcidBzoCas13b crRNA direct repeat sequence780Nucleic AcidPbTCas13b crRNA direct repeat sequence781Nucleic AcidPspCas13b crRNA sequence782Nucleic AcidPspCas13b crRNA direct repeat sequence T17C783Nucleic AcidPspCas13b crRNA direct repeat sequence T18C784Nucleic AcidPspCas13b crRNA direct repeat sequence T19C785Nucleic AcidDMPK CUG Exp786Nucleic AcidCUG exp787Nucleic AcidCAGx9 crRNA788Nucleic AcidCUGx17789Nucleic Acidconserved consensus PAS EXPERIMENTAL EXAMPLES

[0326] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0327] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out certain embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.Example 1: Targeted Cleavage and Polyadenylation of RNA by CRISPR-Cas13

[0328] The data presented herein demonstrates that fusion of catalytically dead Cas13 to a single mammalian polyadenylation factor, Nudix Hydrolase 21 (NUDT21), allows for site-specific CRISPR-Cas13-guided cleavage and polyadenylation of RNA in mammalian cells. This approach is termed Postscriptr and can be utilized for the non-genomic manipulation of gene expression and may have potential future therapeutic applications for treating human RNA processing diseases.

[0329] The 3' site of RNA cleavage and addition of a poly(A) tail is precisely determined by intrinsic polyadenylation signal (PAS) sequences typically composed of a canonical hexamer motif AAUAAA and upstream and downstream sequence elements (USE and DSE, respectively) (Tian & Graber, Wiley Interdiscip Rev RNA, 2012, 3:385-396). The AAUAAA PAS motif is typically found ~25 nucleotides upstream of the RNA cleavage site and is directly bound by two components of the cleavage and polyadenylation specific factor (CPSF) complex: cleavage and polyadenylation factor 30 (CPSF30) and WD repeat-containing protein 33 (WDR33) (Chan et al., Genes Dev, 2014, 28:2370-2380). Components of the cleavage factor Im (CFIm) complex bind directly to the USE motif UGUA which occurs ~50 nucleotides upstream of the RNA cleavage site (Ellkon et al., Nat Rev Genet, 2013, 14:496-506). In transcripts that lack the canonical AAUAAA motif, the CFIm complex functions as the primary determinant of poly(A) signal recognition (Venkataraman et al., Genes Dev, 2005, 19:1315-1327). Nudix Hydrolase 21 (NUDT21 / CPSF5), the RNA binding component of the CFIm complex, functions as an activator of 3' end processing and regulator of alternative polyadenylation site choice (Zhu et al., Mol Cell, 2018, 69:62-74). Binding of the CFIm complex is among the first steps in 3' end processing and functions to recruit additional co-factors to the 3' end processing machinery. Direct interactions between components of the polyadenylation supercomplex and the C-terminal domain of RNA polymerase II trigger the disassembly of the elongation complex and coordinate 3' end processing with transcription termination (Zhao et al., Microbiol Mol Biol Rev, 1999, 63:405-45).

[0330] Cas13 is a bacterial-derived crRNA-guided endonuclease with a specific affinity for RNA (Abudayyeh et al., Science, 2016, 353:aaf5573). Similar to Cas9, mutation of residues within the nuclease domains of Cas13 generates a catalytically dead enzyme but retains RNA binding affinity (dCas13). Recently, fusion of dCas13 to mammalian RNA modifying enzymes has been shown to be useful for manipulating RNA in mammalian cells (Abudayyeh et al., Nature, 2017, 550:280-84; Cox et al., Science, 2017, 358:1019-27; O'Connell, J Mol Biol, 2019, 431:66-87). To direct polyadenylation complex formation using dCas13, three fusion proteins were designed combining the catalytically dead PspCas13b (dPspCas13b) with RNA binding components of the human 3' end processing machinery, including CPSF30, WDR33, and NUDT21 (Figure 1A). An N-terminal 3x FLAG epitope tag was included for protein detection and a long flexible amino acid peptide linker [GGGGSGGGGS (SEQ ID NO:69)] between dPspCas13b and the polyadenylation components to reduce the likelihood of steric hindrance.

[0331] Unlike RNA modifying applications utilizing dCas13 which occur in the cytosol, post-transcriptional cleavage and polyadenylation of RNA occurs within the eukaryotic nucleus and therefore requires efficient nuclear localization of dCas13 fusion proteins. The large size of Cas13 and its lack of intrinsic nuclear localization signals (NLSs) could prevent efficient nuclear localization in mammalian cells. Consistent with this, the dPspCas13b fusion proteins lacking a mammalian NLS were retained in the cytoplasm when expressed in mammalian COS7 cells, as detected by immunocytochemistry using an anti-FLAG antibody (Figure 5). Surprisingly, the addition of a classical SV40 NLS or bipartite NLS from nucleoplasmin (NPM) were both insufficient to promote nuclear localization of the dPspCas13b fusion proteins (Figure 5). However, the addition of a single copy of the non-classical bipartite NLS derived from the yeast Ty1 retrotransposon, which utilizes normal cellular import machinery but contains a linker sequence nearly three times as long as a typical bipartite NLS (McLane et al., NAR, 2008, 36:4317-26; Kenna et al., Mol Cell Biol, 1998, 18:1115-24), resulted in robust nuclear localization of all three dPspCas13b fusion proteins (Figure 1B).

[0332] Next, a reporter gene was designed to detect alternative cleavage and polyadenylation in mammalian cells capable of switching between fluorescent and non-fluorescent open reading frames of superfolder GFP (sfGFP) (Figure 2). sfGFP forms a beta barrel comprised of 11 antiparallel beta strands, which can tolerate sequence insertions between the 10 th< and 11 th< beta strands but loses fluorescence if the 11 th< beta strand is removed (Kamiyama et al., Nat Commun, 2016, 7:11046; Feng et al., Nat Commun, 2017, 8:370). A sfGFP reporter construct was generated with the coding sequence for the 11 th< beta strand embedded within a prototypical mammalian intron (second intron of the rabbit beta globin gene) (sfGFPapa) (Figure 2A). The coding sequence of the 11 th< beta strand was designed in-frame with the upstream sfGFP coding sequence so that translation of the proximal open reading frame would encode a complete sfGFP sequence, albeit with a 14 amino acid linker sequence between the 10 th< and 11 th< beta strand resulting from translation of the intervening intronic sequence [sfGFP(1-10)-L-11]. Expression of the sfGFPapa reporter in mammalian COS7 cells resulted in almost no detectable fluorescence, suggesting efficient removal of the intron containing the 11 th< beta strand and translation of the open reading frame encoding the non-fluorescent sfGFP(1-10) protein (Figure 2B to Figure 2D). However, cells expressing the sfGFPapa reporter treated with the splicing inhibitor isoginkgetin (O'Brien et al., J Biol Chem, 2008, 283:33147-54) resulted in detectable green fluorescence after 24 hours, demonstrating that the modified sfGFP open reading frame containing the linker between the 10 th< and 11 th< beta strands is functional (Figure 6A and Figure 6B). Insertion of an SV40 polyadenylation cassette downstream of coding sequence for the 11 th< beta strand of sfGFP (sfGFPapa-pA) resulted in robust green fluorescence, demonstrating that intronic cleavage and polyadenylation of the sfGFPapa reporter promotes utilization of the functional sfGFP(1-10)-L-11 open reading frame (Figure 2E to 2H). Together, these data demonstrate that the sfGFPapa reporter gene encodes both functional and non-functional open reading frames which can be switched in response to changes in splicing or intronic cleavage and polyadenylation.

[0333] To determine whether the dPspCas13b fusion proteins could promote CRISPR-Cas13-mediated cleavage and polyadenylation of the sfGFPapa reporter mRNA, a crRNA was designed targeting an intronic sequence downstream of the coding sequence of the 11 th< beta strand of sfGFP in the sfGFPapa reporter (Figure 3A). In live cell assays, no fluorescence signal was detected in cells expressing any of the dPspCas13b fusion proteins using a non-targeting crRNA, relative to the sfGFPapa reporter alone (Figure 3B and Figure 7). However, expression of the intron targeting crRNA with the dPspCas13b-NUDT21 fusion protein, but not the CPSF30 or WDR33 fusions proteins, resulted in detectable green fluorescent cells after 24 hours (Figure 3B and Figure 7).

[0334] 3' rapid amplification of cDNA ends (3' RACE) was performed to determine whether the fluorescence signal was due to CRISPR-Cas13-mediated targeted cleavage and polyadenylation of the sfGFPapa reporter mRNA. RACE compatible cDNA was generated from total RNA using a poly(T) oligonucleotide containing two 5' nested primer sequences. Using two nested reporter specific primers upstream of the crRNA target sequence, a high molecular weight band was detected corresponding to the predicted size of the sfGFPapa pre-mRNA transcript for both samples (Figure 3C). Interestingly, in cells targeted with the intronic crRNA, an additional broad band of smaller RACE products was detected, suggesting that cleavage and polyadenylation may have occurred near the targeted crRNA sequence (Figure 3C and Figure 3D). To determine the sequence of the 3' RACE products, the 200-500 base pair region was gel purified and subcloned using TOPO TA cloning. 11 unique clones were isolated and sequenced which revealed that sfGFPapa mRNAs were cleaved and polyadenylated at sites ranging from -7 to +110 nucleotides relative to the 3' side of the crRNA target sequence (Figure 3E). In contrast to mammalian cleavage which occurs primarily after a CA dinucleotide, Postscriptr-induced cleavage most often occurred at a C or T nucleotide (Figure 3F). Interestingly, four of the 11 clones showed a 3' addition of non-templated nucleotides prior to poly (A) tail elongation, which is thought to be rare in mammalian species but prevalent in plant mRNAs (Jin & Bian, RNA, 2004, 10:1695-97). These data demonstrate that the dPspCas13b-NUDT21 fusion protein was sufficient to induce cleavage and polyadenylation of a reporter mRNA at an intronic sequence targeted by a crRNA.

[0335] To determine whether Postscriptr could promote alternative cleavage and polyadenylation of an endogenously expressed human mRNA, transcripts encoding the human sterol regulatory element binding protein 1 (SREBP1) were targeted. SREBP1 is a ubiquitously expressed transcription factor which transactivates genes that contain sterol regulatory eleme...

Claims

1. A fusion protein comprising: a) a CRISPR-associated (Cas) protein; and b) a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21.

2. The fusion protein of claim 1, wherein the Cas protein is catalytically dead Cas13 (dCas13).

3. The fusion protein of claim 2, wherein dCas13 comprises SEQ ID NO: 47, or a variant thereof.

4. The fusion protein of claim 1, wherein NUDT21 comprises SEQ ID NO: 51, or a variant thereof.

5. The fusion protein of any of claims 1-4, wherein the fusion protein further comprises a nuclear localization signal (NLS).

6. The fusion protein of claim 5, wherein NLS comprises SEQ ID NO: 75, or a variant thereof.

7. The fusion protein of any of claims 1-6, wherein the fusion protein comprises SEQ ID NO: 698, or a variant thereof.

8. A nucleic acid molecule encoding a fusion protein of any of claims 1-7.

9. An in vitro method of modulating the cleavage, polyadenylation or both of an RNA transcript in a cell, the method comprising administering to the cell: a fusion protein of any of claims 1-7 or the nucleic acid molecule of claim 8 and a guide nucleic acid comprising a sequence complimentary to a target RNA sequence in the RNA transcript.

10. A fusion protein according to any of Claims 1-7 or the nucleic acid molecule of Claim 8, and a guide nucleic acid comprising a sequence complimentary to a target RNA sequence in the RNA transcript, for use in in vivo modulation of the cleavage, polyadenylation or both of an RNA transcript in a subject.

11. The fusion protein of any of claims 1-7 or the nucleic acid molecule of claim 8, for use as a medicament.

12. The fusion protein of any of claims 1-7 or the nucleic acid molecule of claim 8 for use in the treatment of human RNA processing diseases.