Targeted nuclear RNA cleavage and polyadenylation using CRISPR-Cas

Abstract

The present invention provides proteins, nucleic acids, systems and methods for visualizing or modulating RNA.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This 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, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Post-transcriptional cleavage and polyadenylation of messenger and long non-coding RNA are coordinated by a supercomplex of about 20 individual proteins in 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 important role in controlling RNA transcription stability, nuclear export and translation efficiency (Colgan and Manley, Genes Dev, 1997, 11: 2755-66; Guhaniyogi and Brewer, Gene, 2001, 265: 11-23; Wu and Brewer, Gene, 2012, 500: 10-21; Carmody and Wente, J Cell Sci, 2009, 122: 1933-37). More than half of all human RNA transcripts contain multiple polyadenylation signal sequences, which can undergo selective cleavage and polyadenylation during development and cell differentiation (Tian and Manley, Nat Rev Mol Cell Biol, 2017, 18: 18-30; Elkon et al., Nat Rev Genet, 2013, 14: 496-506). Selective cleavage and polyadenylation are important mechanisms for controlling gene expression, and defects in 3' end processing can cause numerous human diseases (Chang et al., Endocrinol Metab, 2017, 32: 413-21; Curinha et al., Nucleus, 2014, 5: 508-19).

[0004] Therefore, there is a need in the art for compositions and methods for regulating selective cleavage and polyadenylation. The present invention satisfies this need. Summary of the invention

[0005] In one aspect, the present invention provides a fusion protein comprising (a) a crisper-associated (Cas) protein; and (b) a cleavage and / or polyadenylation protein. In one embodiment, the Cas protein is a catalytically dead Cas13 (dCas13). In one embodiment, dCas13 comprises a sequence selected from SEQ ID NO: 47-48 or a variant thereof. In one embodiment, the cleavage or polyadenylation protein is NUDT21. In one embodiment, NUDT21 comprises a sequence selected from SEQ ID NO: 51-58 or a variant thereof.

[0006] In one embodiment, the fusion protein further comprises a nuclear localization signal (NLS). In one embodiment, the NLS comprises a sequence selected from SEQ ID NO: 75-695 or a variant thereof. In one embodiment, the fusion protein comprises a sequence selected from SEQ ID NO: 696-698 or a variant thereof.

[0007] In one embodiment, the invention provides a method of regulating cleavage, polyadenylation, or both of an RNA transcript in a subject. In one embodiment, the method comprises administering to the subject: a fusion protein of the invention or a nucleic acid molecule encoding the method of the invention and a guide nucleic acid comprising a sequence complementary to a target RNA sequence in the RNA transcript. In one embodiment, the method is an in vitro method. In one embodiment, the method is an in vivo method.

[0008] In one aspect, the present invention provides a fusion protein comprising (a) a crisper-related (Cas) protein; and (b) a fluorescent protein. In one embodiment, the Cas protein is a catalytically dead Cas13 (dCas13). In one embodiment, dCas13 comprises a sequence selected from SEQ ID NO: 47-48 or a variant thereof. In one embodiment, the fluorescent protein is selected from the group consisting of: eGFP, mCherry, sfGFP, sfGFP (1-10), sfGFP (1-10) -L- (11) and 7xS11. In one embodiment, the fluorescent protein comprises a sequence selected from SEQ ID NO: 59-66 or a variant thereof.

[0009] In one embodiment, the fusion protein further comprises a nuclear localization signal (NLS). In one embodiment, the NLS comprises a sequence selected from SEQ ID NO: 5-695 or a variant thereof. In one embodiment, the fusion protein comprises a sequence selected from SEQ ID NO: 699-701 or a variant thereof.

[0010] In one embodiment, the present invention provides a method for visualizing nuclear RNA of a subject. In one embodiment, the method comprises administering to the subject: a fusion protein of the present invention or a nucleic acid molecule encoding the method of the present invention and a guide nucleic acid comprising a sequence complementary to a target RNA sequence in the nuclear RNA transcript; and visualizing the nuclear RNA. In one embodiment, the method is an in vitro method. In one embodiment, the method is an in vivo method.

[0011] In one aspect, the present invention provides a fusion protein comprising (a) a crisper-related (Cas) protein; and (b) an NLS. In one embodiment, the Cas protein is Cas13. In one embodiment, Cas13 comprises a sequence selected from SEQ ID NO: 1-46 or a variant thereof. In one embodiment, the NLS comprises a sequence selected from SEQ ID NO: 75-695 or a variant thereof. In one embodiment, the fusion protein comprises a sequence of SEQ ID NO: 702 or a variant thereof.

[0012] In one embodiment, the invention provides a method of reducing the amount of nuclear RNA or cleaved nuclear RNA in a subject. In one embodiment, the method comprises administering to the subject: a fusion protein of the invention or a nucleic acid molecule encoding the method of the invention and a guide nucleic acid comprising a sequence complementary to a target RNA sequence in the nuclear RNA transcript. In one embodiment, the method is an in vitro method. In one embodiment, the method is an in vivo method.

[0013] In one embodiment, the present invention provides a nucleic acid encoding the fusion protein of the present invention.

[0014] In one embodiment, the present invention provides the use of the fusion protein of the present invention or the nucleic acid molecule of the present invention in the manufacture of a medicament. In one embodiment, the present invention provides the use of the fusion protein of the present invention or the nucleic acid molecule of the present invention in the treatment of a disease or condition associated with RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 ,include Figure 1 A and Figure 1 B, depicts the design and expression of a fusion protein encoding catalytically dead Cas13 and human polyadenylation complex factors. Figure 1 A depicts a diagram of a vector encoding a fusion between the 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 detecting nuclear localization of dPspCas13b fusion protein expressed in mammalian COS7 cells using primary anti-FLAG antibody and Alexa488-conjugated secondary antibody. Scale bar in B, 10 μm.

[0016] Figure 2 ,include Figure 2 A to Figure 2 H, Depicts the design and generation of fluorescent reporters for RNA cleavage and polyadenylation in mammalian cells. Figure 2 A Schematic diagram depicting the sfGFPapa reporter gene plasmid. Figure 2 B depicts the green fluorescent protein superfolded GFP (sfGFP), which forms a β barrel consisting of 11 β strands. Removal of the 11th β strand abolishes the fluorescence. Figure 2 C depicts transcription of the sfGFPapa reporter gene and processing of the resulting transcript that removes the coding sequence for the 11th sfGFP β chain. Figure 2 D demonstrates that removal of the 11th β-strand results in a lack of fluorescence signal in mammalian cells. Figure 2 E depicts a map of the sfGFPapa-pA reporter plasmid, which contains an upstream polyadenylation sequence. Figure 2 F is a schematic diagram depicting that sfGFP can tolerate a linker sequence between the 10th and 11th beta strands without abolishing fluorescence. Figure 2 G depicts transcription of the sfGFPapa-pA reporter gene and processing of the resulting transcript. Figure 2 H demonstrates that the processed transcript produces strong green fluorescence due to the translation of the sfGFP(1-10)-L-(11) functional open reading frame. Figure 2 D and Figure 2 Scale bar in H, 100 μm.

[0017] Figure 3 ,include Figure 3 A to Figure 3 F, Depiction of CRISPR-Cas13-mediated cleavage and polyadenylation of reporter mRNA in mammalian cells. Figure 3 A depicts a diagram showing the Postscriptr targeting strategy to induce selective cleavage and polyadenylation of the sfGFPapa reporter construct. The crRNA was designed to target an intronic sequence downstream of the coding sequence of the 11th beta strand of sfGFP. Figure 3 B depicts the expression of dPspCas13b-NUDT21 fusion protein and demonstrates that intron-targeted crRNA produced green fluorescent cells after 24 h. Figure 3C depicts 3′ RACE products amplified from cells expressing dPspCas13b-NUDT21 fusion protein and non-targeting control or intron-targeting crRNA. Figure 3 D and Figure 3 E depicts the sequences of the five most proximal 3' RACE products relative to the crRNA target site. For a complete list of 3' RACE sequences, see Example 3. Green underlined and lowercase nucleotides highlight 3' non-templated nucleotide additions. Figure 3 F depicts the nucleotide frequencies of PostscriptR-mediated cleavage and polyadenylation sites.

[0018] Figure 4 ,include Figure 4 A to Figure 4 I, depicts PostscriptR-mediated alternative cleavage and polyadenylation of human SREBP1 in HEK293T cells. Figure 4 A. Diagram depicting the SREBP1 maturation pathway. Figure 4 B depicts that the human SREBP1 locus contains an intronic PAS between exon 7 and exon 8, which results in the translation of the SREBP1Δ isoform that terminates translation adjacent to the normal S2P cleavage site. Figure 4 C depicts the SREP1 sequence and the SREBP1Δisoform sequence. Figure 4 D Diagram depicting the PostscriptR-mediated targeting strategy and the sequences of recovered PostscriptR-induced cleavage transcripts. Figure 4 E Diagram depicting the PostscriptR-mediated targeting strategy and the sequences of recovered PostscriptR-induced polyadenylated transcripts. Figure 4 F depicts predicted translation termination. Figure 4 G depicts quantitative real-time PCR (qRT-PCR) gene expression analysis of SREBP1 transcript levels upstream of crRNA target sequence in HEK293T cells targeted with non-targeting or SREBP1-targeting crRNA. Figure 4 H depicts quantitative real-time PCR (qRT-PCR) gene expression analysis of SREBP1 transcript levels across the crRNA target sequence in HEK293T cells targeted with non-targeting or SREBP1-targeting crRNA. Figure 4 I depicts quantitative real-time PCR (qRT-PCR) gene expression analysis of transcript levels of the LDLR gene in HEK293T cells targeted with non-targeting or SREBP1-targeting crRNA.

[0019] Figure 5Depicts experimental results demonstrating that the classical mammalian nuclear localization signal is insufficient to promote nuclear localization of the dPspCas13b fusion protein. Immunohistochemistry using a primary anti-FLAG antibody and a secondary Alexa488-conjugated secondary antibody was used to detect the localization of the dPspCas13b fusion protein expressed in mammalian COS7 cells. The fusion protein does not contain NLS, the classical SV40 NLS, or a bipartite NLS from nucleoplasmin (NPM). Scale bar = 10 μm

[0020] Figure 6 ,include Figure 6 A and Figure 6 B, depicts experimental results demonstrating that inhibition of splicing promotes expression of the sfGFP(1-10)-L-(11) open reading frame. Figure 6 A Diagram depicting the sfGFPapa reporter gene and the predicted transcription and processing steps resulting from treatment with the splicing inhibitor isoginkgetin. Figure 6 B depicts COS7 cells transiently transfected with the sfGFPapa reporter gene for 24 hours were treated with DMSO or isoginkgetin for an additional 24 hours. Cells treated with isoginkgetin produced detectable green fluorescence. Scale bar in B = 100 μm.

[0021] Figure 7 ,include Figure 7 A and Figure 7 B, depicts experimental results demonstrating that fusions of dPspCas13b with CPSP30 or WDR33 are insufficient to promote cleavage and polyadenylation of the sfGFPapa reporter mRNA. Figure 7 A depicts Postscriptr targeting of the sfGFPapa reporter gene using the dPspCas13b-CPSF30 fusion protein using intron-targeting crRNA did not produce detectable green fluorescence relative to the control crRNA. Figure 7 B depicts Postscriptr targeting of the sfGFPapa reporter gene using the dPspCas13b-WDR33 fusion protein using intron-targeting crRNA did not produce detectable green fluorescence relative to the control crRNA. Scale bar = 100 μm

[0022] Figure 8 Figure 3. Experimental results demonstrating Postscriptr-mediated selective cleavage and polyadenylation of human SREBP1 transcripts. PCR amplified 3' RACE products from cells expressing dPspCas13b-NUDT21 with either a non-targeting control or SREBP1-targeting crRNA.

[0023] Fig. 9 ,include Fig. 9 A to Fig. 9 B, depicts experimental results demonstrating that Cas13b crRNA sequence modification enhances Postscriptr activity. Fig. 9 A depicts a schematic diagram demonstrating that CRISPR-Cas13 guide RNAs are typically expressed in mammalian cells from a polymerase III (PolIII) promoter terminated by a poly (T) sequence. It has recently been shown that a stretch of only 4 Ts can result in a 75% reduction in full-length expression of small RNAs from the U6 promoter. Fig. 9 B depicts a schematic diagram demonstrating that multiple Cas13b crRNAs contain direct repeat (DR) sequences. Fig. 9 C depicts a schematic diagram demonstrating that the DR sequence contains segments of 4 or 5 Ts. Fig. 9 D depicts the crystal structure demonstrating that these nucleotides fall within the loop region and that some positions do not make direct molecular interactions. Yellow highlighting in 9B and yellow arrows in 9D. Fig. 9 E depicts a schematic diagram demonstrating the generation of conservative mutations (T to C) in the DR of PspCas13b crRNA. Fig. 9 F depicts experimental results demonstrating determination of their relative effectiveness in Postscriptr-targeted activation of the sfGFPapa fluorescent reporter gene. Notably, two mutations, T17C and T18C, resulted in enhanced Postscriptr activity, while another mutation, T19C, resulted in reduced activity, similar to a non-targeting (NT) guide RNA.

[0024] Fig.10 Depicted is a sequence alignment of NUDT21 proteins from human, fly, and worm.

[0025] Fig.11Depict experimental results demonstrating the relative activity of Postscriptr fusion proteins. The effects of different sequence modifications on dPspCas13b-NUDT21 fusion proteins were evaluated using activation of the sfGFPapa reporter gene in mammalian cells guided by intron-targeted crRNA. Notably, fly and worm orthologs of NUDT21 showed comparable activation levels relative to humans, likely due to their high sequence conservation across species and conservative role in polyadenylation. The N-termini of these orthologs are different, which is not only dispensable for Postscriptr-mediated activation, but also produces enhanced activity. This may be due to the fact that acetylation of lysine in these regions has been shown to inhibit NUDT21 activity. Mutations in residues that prevent NUDT21 from binding to RNA (R63S and F103A) have little effect on its function as a fusion with dCas13. Remarkably, tandem dimers of NUDT21, which usually function as obligate dimers, produce significantly enhanced activation. In addition, Postscriptr enzymes with a truncated C-terminus of dPspCas13 (Δ984-1090) showed similar levels of sfGFPapa activation.

[0026] Fig.12 A schematic diagram illustrating the molecular genetic basis of myotonic dystrophy type 1 (DM1) is depicted. DM1 is a monogenic autosomal dominant genetic disease characterized by progressive muscle atrophy, myotonia, arrhythmias and cognitive dysfunction. DM1 is the most common adult-onset muscular dystrophy and is caused by the amplification and expression of CUG trinucleotide repeats in the non-coding 3' untranslated region of the human myotonic dystrophy protein kinase (DMPK) gene. Mutant DMPK mRNA with more than about 50 CUG repeats forms toxic nuclear RNA foci that prevent normal DMPK expression and induces a wide range of defects in alternative splicing and selective polyadenylation by sequestering members of the blind muscle-like (MBNL) family of RNA-binding proteins. There is no approved therapy specific for DM1, and current strategies targeting CUG RNA repeats cannot address the loss of DMPK expression.

[0027] Fig.13 ,include Fig.13 A to Fig.13D, depicts therapeutic correction of DM1 with targeted selective cleavage and polyadenylation of human DMPK transcripts. Selective polyadenylation (APA) of RNA is an important regulatory mechanism controlling gene expression during development and disease. Recent deep sequencing revealed that some DMPK transcripts can be selectively cleaved and polyadenylated at a position upstream of the CUG amplification site, suggesting that manipulation of APA may be a useful approach for retaining DMPK expression while preventing transcription of downstream toxic repeat RNAs. Fig.13 A Schematic diagram depicting Postscriptr. Fig.13 B depicts a schematic demonstrating that Postscriptr combines the programmable RNA targeting capabilities of CRISPR-Cas13 with mammalian polyadenylation factors to induce site-specific cleavage and polyadenylation of RNA transcripts. Fig.13 C depicts experimental results demonstrating that Postscriptr can robustly induce selective cleavage and polyadenylation of endogenous human DMPK transcripts upstream of the CUG repeat expansion site. Fig.13 D depicts experimental results demonstrating that targeted selective polyadenylation of mutant DMPK transcripts by Postscriptr can rescue DMPK expression and prevent transcription of downstream toxic CUG repeat RNA.

[0028] Fig.14 ,include Fig.14 A to Fig.14 C, Delineation of the molecular origin of DM1. Fig.14 A depicts a schematic diagram demonstrating that myotonic dystrophy type 1 (DM1) is caused by a microsatellite CTG repeat expansion in the 3'UTR of the human DMPK gene. Fig.14 B Describing DMPK CUG exp The RNA forms a stable hairpin that is retained in nuclear foci, thereby preventing normal DMPK gene expression and sequestering the MBNL family of RNA-binding proteins. Fig.14 C depicts a schematic diagram demonstrating that nuclear foci lead to widespread defects in alternative splicing and polyadenylation.

[0029] Fig.15 ,include Fig.15 A to Fig.15 C, Schematic diagram depicting selective polyadenylation. Fig.15 A depicts a schematic demonstrating that deep sequencing of the human transcriptome reveals that more than half of all genes undergo alternative cleavage and polyadenylation at intronic, proximal, or distal polyadenylation signals (PAS). Fig.15B depicts a schematic demonstrating that a supercomplex of approximately 20 proteins coordinates 3' end processing and transcription termination. The RNA binding components of the CFIm and CPSF subcomplexes recognize RNA motifs in the pre-RNA transcript that specify sites for cleavage and polyadenylation. Fig.15 C depicts a schematic diagram demonstrating that the novel RNA editing technology described herein, termed Postscriptr, is sufficient to induce targeted cleavage and polyadenylation using CRISPR-Cas13.

[0030] Fig.16 ,include Fig.16 A and Fig.16 B, Depicts experimental results demonstrating that nuclear localization of dCas13 fusion proteins requires a unique NLS. Fig.16 A Schematic diagram depicting the vector-encoded fusion between catalytically dead PspCas13b and NUDT21. Fig.16 B depicts experimental results demonstrating that mammalian cleavage and polyadenylation occur in the nucleus. The non-canonical nuclear localization signal (NLS) from the yeast retrotransposon Ty1 is essential for the nuclear localization of the dPspCas13b-NUDT21 fusion protein.

[0031] Fig.17 ,include Fig.17 A and Fig.17 B, depicts the results of experiments demonstrating targeted cleavage and polyadenylation of RNA. Fig.17 A depicts a schematic diagram showing the Postscriptr targeting strategy to induce selective cleavage and polyadenylation of the sfGFPapa reporter construct. The crRNA was designed to target an intronic sequence downstream of the coding sequence of the 11th beta strand of sfGFP. Fig.17 B depicts the expression of dPspCas13b-NUDT21 fusion protein and intron-targeted crRNA resulting in green fluorescent cells after 24 h.

[0032] Fig.18 ,include Fig.18 A and Fig.18 B, depicts the results of experiments demonstrating Postscript editing of endogenous human DMPK transcripts. Fig.18 A depicts the design of a guide RNA targeting the DMPK 3'UTR at a position downstream of the DMPK stop codon and upstream of the CUGexp site. Fig.18B depicts the results of experiments demonstrating Postscriptr editing of DMPK transcripts in HEK293T cells, revealing that Postscriptr promotes the utilization of the proximal PAS and reduces the expression of the distal PAS with the full-length DMPK protein coding sequence, as indicated by the retention of the distal exon. Thus, this strategy may provide a mechanism to promote DMPK expression and prevent downstream expression of CUGexp RNA.

[0033] Fig.19 ,include Fig.19 A to Fig.19 D, depicts the results of an experiment demonstrating targeting of CUG repeat RNA foci with dCas13. Fig.19 A. Schematic diagram depicting the fusion of eGFP to dPspCas13b to visualize the subcellular localization of dCas13. This construct was named HiLightr Green. Fig.19 B depicts DT960, an expression plasmid containing 960 CUG repeats in the context of human DMPK exons 11-15, which was used to induce nuclear foci. Fig.19 C Schematic diagram of the experiment depicting the CAGx9 guide RNA. Fig.19 D depicts experimental results demonstrating that HiLightr green colocalizes with mCherry-MBNL1 to RNA foci when targeted with antisense CAGx9 guide RNA but remains unlocalized when a non-targeting guide RNA is used.

[0034] Fig. 20 ,include Fig. 20 A and Fig. 20 B, depicts experimental results demonstrating that CUGexp RNA blocks mRNA expression. Fig. 20 A depicts luciferase expression vectors containing human DMPK 3'UTR with 12 CUG repeats (pGL3P-DT12a) or 960 CUG repeats (pGL3P-DT960). Fig. 20 B depicts experimental results demonstrating that the presence of 960 CUG repeats resulted in a 90% reduction in luciferase activity when expressed in COS7 cells.

[0035] Fig.21 ,include Fig.21 A to Fig.21 D, depicts an inducible DM1 mouse model. An inducible DM1 humanized mouse model was used that expresses 960CUG amplified RNA in the context of human DMPK exons 11-15. Fig.21 A depicts a schematic diagram showing the cross between the transgene and the skeletal muscle specific rtTA transgene. Fig.21B depicts a schematic diagram demonstrating that transgene expression can be induced by doxycycline (dox). Fig.21 C depicts the PostscriptR editing components encoded in the lentiviral vector. Fig.21 D depicts the use of lentiviral vectors to generate lentiviral particles that are delivered concomitantly with dox during postnatal development. PostscriptR editing of CUG-encoded transcripts is used to model the effectiveness of postscriptR mitigation of DM1.

[0036] Fig. 22 ,include Fig. 22 A to Fig. 22 D, depicts experimental results demonstrating the development of a robust nuclear-localized CRISPR-Cas13 fusion protein for visualization of toxic RNA foci. Fig. 22 A depicts the design of a catalytically dead PspCas13b (dPspCas13b) encoding N-terminal 3xFLAG and Ty1 NLS and C-terminal eGFP. F–3xFLAG epitope; NLS–Ty1 nuclear localization sequence; pA–SV40 polyadenylation sequence. Fig. 22 B depicts a diagram describing the components of the DT960 vector encoding the C-terminal genomic fragment of human DMPK (exons 11-15) with 960 CTG repeat expansions. Fig. 22 C depicts the design of CAGx9 crRNA and its use CUG exp Predicted targeting by RNA. Fig. 22 D depicts a display showing expression of CUG exp Representative images of cellular localization of hilightR green in COS7 cells targeted with non-targeting or CAGx9 crRNA. Scale bar, 10 μm.

[0037] Fig.23 ,include Fig.23 A and Fig.23 B, depiction of hilightR green and CUG exp Experimental results of co-localization of foci and MBNL1. Fig.23 A depicts immunohistochemistry using anti-FLAG antibody for detection of hilightR red, which, when targeted with CAGx9 crRNA, is identical to CUG detected using FISH. exp RNA colocalization. Fig.23 B depicts the expression of CUG when targeted with CAGx9 crRNA instead of non-targeting crRNA. exp HilightR green co-localized with mCherry-MBNL1 in COS7 cells at RNA foci. Scale bar, 10 μm.

[0038] Fig.24 ,include Fig.24 A and Fig.24 B, depicts experimental results demonstrating degradation of toxic RNA foci by CRISPR-Cas13. Fig.24 A depicts co-expression of active PspCas13b encoding Ty1NLS (eraseR) significantly reduced expression of CUG when targeted with CAG crRNA designed with target sequence in all three frameworks. exp The number of RNA foci in the cells was detected by mCherry-MBNL1. Fig.24 B depicts a representative micrograph of cells targeted by eraseR, showing foci detected by mCherry-MBNL1 that are significantly reduced in number and appear weaker. Scale bar, 10 μm. **=p value < 0.01, ***=p value < 0.001, ****=p value < 0.0001.

[0039] Fig.25 ,include Fig.25 A to Fig.25 C, depicts the detection of inducible CUG in COS7 cells. exp Experimental results of RNA foci. Fig.25 A depicts the induction of RNA foci in COS7 cells expressing 960 copies of CUG repeats as detected using FISH with a CAG repeat antisense probe. AF488–Alexa Fluor 488. Fig.25 B depicts CUG exp Expression of the RNA induced MBNL1 localization to foci, as detected using mCherry-MBNL1 fusion protein. Fig.25 C depicts the expression of CUG exp Localization of dPspCas13b-mCherry (hilightR red) in COS7 cells guided by non-targeting or CAGx9 crRNA. Scale bar, 10 μm.

[0040] Fig.26 Depicts experimental results demonstrating colocalization of hilightR green with splicing speckles. Consistent with previous reports, CUG was detected using anti-SC-35 antibodies. exp RNA foci labeled by HiLightR green, targeted by CAGx9 crRNA, colocalize with splicing speckles. Scale bar, 10 μm.

[0041] Fig. 27 Depicts experimental results demonstrating that catalytically dead Cas13 (dCas13) does not significantly reduce CUGexp Expression of dPspCas13b targeted with CAGx9 crRNA did not significantly reduce the number of CUG foci per cell, as detected by mCherry-MBNL1. exp Number of RNA foci. ns-not significant.

[0042] Fig.28 ,include Fig.28 A to Fig.28 D, depicts a diagram showing therapeutic modulation of DM1 by CRISPR-Cas13. Fig.28 A depicts myotonic dystrophy type 1 is caused by the expansion and expression of a CUG repeat sequence in the 3' noncoding UTR of the human DMPK gene. This CUG expansion forms a stable hairpin structure that binds and sequesters the MBNL family of RNA-binding proteins, leading to widespread defects in alternative splicing and polyadenylation. Fig.28 B depicts that the CUG repeat sequence is resistant to cleavage induced by antisense oligonucleotides (ASOs), however, ASOs have been successfully used to block the binding of MBNL1 protein. However, there are still many challenges in delivering therapeutically effective levels of ASOs to human tissues. Fig.28 C depicts specific binding of crRNA or possibly crRNA alone to the dCas13 guide can be used to block MBNL protein and rescue splicing and polyadenylation defects, or to highlight CUG repeat RNA foci when combined with fluorescent proteins. Fig.28 D depicts that 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 RNA-dependent translation of toxic peptides.

[0043] Fig.29Depicted is a schematic diagram showing that myotonic dystrophy type 1 (DM1) is an inherited multisystem, progressively debilitating disease that occurs in 1 / 8,000 individuals, with an incidence as high as 1 / 500 in certain populations. Cardiac complications occur in approximately 80% of DM1 patients and are the leading cause of death. DM1 is caused by the expansion and expression of CUG trinucleotide repeats in the noncoding 3' untranslated region of the human myotonic dystrophy protein kinase (DMPK) gene. Mutant DMPK mRNA with greater than about 50 CUG repeats forms toxic nuclear RNA foci that prevent normal DMPK expression and induce extensive defects in alternative splicing by sequestering members of the myocardial blind (MBNL) family of RNA binding proteins. Due to the large number of disrupted muscle genes underlying the pathogenesis of DM1, patients often exhibit a variety of clinical cardiac phenotypes, including atrial and ventricular arrhythmias, dilated cardiomyopathy, and myocardial fibrosis. RNA-binding CRISPR-Cas13, when targeted with robust non-canonical nuclear localization signals (hilightR and eraseR), can be used to visualize and degrade toxic nuclear RNA foci in cells.

[0044] Fig.30 ,include Fig.30 A to Fig.30 E, depicts results of experiments demonstrating therapeutic rescue of cardiac function in the DM1 mouse model. Fig.30 A depicts the generation of the CUG960 cardiac DM1 mouse model. Fig.30 B depicts the generation of the CUG960 cardiac DM1 mouse model. Fig.30 C depicts a diagram of the eraseR AAV construct. Fig.30 D depicts experimental results demonstrating cardiac-specific gene delivery and expression using AAV9. Fig.30 E depicts delivery of eraseR AAV targeting CUGexp RNA reversal of cellular and electrical abnormalities in DM1 hearts.

[0045] Fig.31 ,include Fig.31 A to Fig.31 D, depicts the results of an experiment demonstrating activation of calcineurin signaling using Postscriptr. Fig.31 A depicts a schematic diagram showing that calcineurin is a Ca2+ / calmodulin-activated protein phosphatase that is autoinhibited by a C-terminal autoinhibitory domain in the absence of calcium signaling. Fig.31 A depicts a schematic diagram showing that upon activation, calcineurin dephosphorylates NFAT transcription factors, which allows nuclear entry and activation of NFAT target genes. Fig.31A depicts a schematic diagram showing the design of guide RNAs for inducing selective cleavage and polyadenylation of the calcineurin (PPP3CB) gene to allow expression of an N-terminal fragment of the calcineurin catalytic domain lacking a C-terminal autoinhibitory domain. Fig.31 A depicts Postscriptr expression in mouse fibroblasts targeting the PPP3CB gene results in nuclear localization of the NFAT-GFP reporter, which normally remains in the cytoplasm and is unaffected by non-targeting guide RNA.

[0046] Fig.32 ,include Fig.32 A to Fig.32 D, Depicts the strategy for enhancing RNA visualization and fusion protein localization using dCas13. Fig.32 A depicts a schematic diagram depicting a fusion of a single green fluorescent protein (GFP) and a catalytically inactive Cas13 (dCas13), which can be used for specific visualization of nuclear RNA repeat foci in cells. Fig.32 A depicts a schematic diagram depicting that the fluorescence complementation inherent in fluorescent proteins (e.g., GFP, superfolded GFP, or superfolded Cherry) can be used to recombinate fluorescent proteins into dCas13 (e.g., complement pair sfGFP 1-10 and sfGFP11). Fig.32 C depicts a schematic depicting that tandem assembly of small non-fluorescent components can be used to recombinant large tandem arrays of fluorescent proteins into dCas13, which has the potential to increase the signal-to-noise ratio of dCas13 targeting RNA. Fig.32 A depicts a schematic diagram depicting that this approach is also useful for targeting a fusion protein (protein 'X') when co-expressed as a fusion with a complementary fluorescent fusion protein (eg, sfGFP1-10).

[0047] Fig.33 ,include Fig.33 A to Fig.33 C, depicts structure-function analysis of Postscriptr RNA editing. Fig.33 A depicts the structural modeling of dCas13b-NUDT21 fusion protein and crRNA using the high-resolution crystal structures of Cas13b (6DTD) and NUDT21 (3MDG). Fig.33 B depicts results demonstrating that NUDT21 forms a native homodimer, which can be expressed as a tandem dimer fused to dCas13b (dCas13b-tdNUDT21) due to the close proximity of the N and C termini. Fig.33C depicts a model depicting the structural orientation of a dCas13b-NUDT21 fusion protein antisense to a target RNA. The orientation of NUDT21 is predicted to occur 3' of the crRNA target sequence on the target RNA, which is consistent with the observed position of postscriptr-induced RNA cleavage and polyadenylation. DETAILED DESCRIPTION

[0048] In one aspect, the present invention is based on the development of new fusion proteins that allow targeted RNA cleavage and polyadenylation of RNA transcripts by CRISPR-Cas. This fusion protein (referred to herein as Postscriptr) comprises a Cas protein, a nuclear localization signal (NLS), and a cleavage and / or polyadenylation protein, such as NUDT21. Mutations in Cas13 produce catalytic dead enzymes (dCas), but retain RNA binding affinity. Therefore, fusions of dCas13 with cleavage and / or polyadenylation proteins allow targeting of cumulative and / or polyadenylation proteins. Postscripter allows non-genomic manipulation of gene expression and can be used for basic research and therapeutic applications.

[0049] Therefore, in one embodiment, the present invention provides compositions and methods for regulating the cleavage, polyadenylation or both of RNA transcripts in a subject. In one embodiment, the present invention provides a fusion protein comprising a CRISPR-related (Cas) protein, a cleavage and / or polyadenylation protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a joint. In one embodiment, the joint connects the Cas protein and the cleavage and / or polyadenylation protein. In one embodiment, the fusion protein comprises a tag.

[0050] In one embodiment, the fusion protein comprises an amino acid sequence that is 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.

[0051] In one embodiment, the invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a cleavage and / or polyadenylation protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a joint. In one embodiment, the joint connects the Cas protein and the cleavage and / or polyadenylation protein. In one embodiment, the fusion protein comprises a tag.

[0052] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is 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.

[0053] The present invention is also based on the unexpected development of a novel fusion protein that allows targeted nuclear RNA cracking and degradation. This fusion protein (referred to herein as EraseR) includes Cas protein and nuclear localization signal (NLS). The CRISPR-Cas13 system is only combined with RNA, and acts as a specific endonuclease to crack the target RNA, bypassing the germline editing risk associated with DNA-bound CRISPR-Cas endonucleases. However, due to their large size and lack of intrinsic positioning signals, Cas13 fusion proteins cannot be effectively positioned to mammalian cell nuclei. EraseR fusion proteins are effectively and efficiently delivered to the nucleus, thereby allowing targeted nuclear RNA cracking and degradation. Therefore, EraseR allows non-genomic manipulation of gene expression, which can be used for basic research and therapeutic applications.

[0054] Therefore, in one embodiment, the present invention provides compositions and methods for reducing the amount of nuclear RNA in a subject.In one embodiment, the present invention provides a fusion protein comprising a Cas protein and an NLS.

[0055] In one embodiment, the fusion protein comprises an amino acid sequence that is 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.

[0056] In one embodiment, the present invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and an NLS.

[0057] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is 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 NO:723.

[0058] The present invention is also based on the unexpected development of a novel fusion protein that allows specific visualization of nuclear RNA. This fusion protein (referred to herein as HilightR) includes a Cas protein and a fluorescent protein. Mutations in Cas13 produce catalytic dead enzymes (dCas), but retain RNA binding affinity. Therefore, the fusion of dCas13 and fluorescent protein allows targeted visualization of RNA. Therefore, HilightR allows visualization of RNA, including nuclear RNA.

[0059] The CRISPR-Cas13 system binds only to RNA and acts as a specific endonuclease to cleave target RNA, bypassing the germline editing risks associated with DNA-binding CRISPR-Cas endonucleases. However, due to their large size and lack of intrinsic localization signals, Cas13 fusion proteins cannot be effectively localized to the mammalian cell nucleus. EraseR fusion proteins are effectively and efficiently delivered to the cell nucleus, allowing targeted nuclear RNA cleavage and degradation. Therefore, EraseR allows non-genomic manipulation of gene expression and can be used for basic research and therapeutic applications.

[0060] In one embodiment, the invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a fluorescent protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a joint. In one embodiment, the joint connects the Cas protein and the fluorescent protein. In one embodiment, the fusion protein comprises a tag.

[0061] In one embodiment, the fusion protein comprises an amino acid sequence that is 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.

[0062] In one embodiment, the invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Cas protein and a fluorescent protein. In one embodiment, the fusion protein further comprises a nuclear localization signal. In one embodiment, the fusion protein further comprises a joint. In one embodiment, the joint connects the Cas protein and the fluorescent protein. In one embodiment, the fusion protein comprises a tag.

[0063] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is 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.

[0064] definition

[0065] 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.

[0066] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

[0067] 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 in this document.

[0068] The nomenclature used herein and the laboratory procedures utilized in analytical chemistry and organic syntheses described below are those well known and commonly employed in the art. Standard techniques or their modifications were used for chemical syntheses and chemical analyses.

[0069] The terms "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 the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0070] As used herein, "about" when referring to a measurable value (such as an amount, a duration, etc.) is meant to encompass deviations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such deviations are appropriate for performing the disclosed methods.

[0071] "Antisense" refers in particular to a nucleic acid sequence of a non-coding strand of a double-stranded DNA molecule encoding a protein, or a sequence 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. An antisense sequence need not be complementary only to the coding portion of the coding strand of a DNA molecule. An antisense sequence may be complementary to a specified regulatory sequence on the coding strand of a DNA molecule encoding a protein, and the regulatory sequence controls the expression of the coding sequence.

[0072] "Disease" is a health condition in an animal wherein the animal is unable to maintain homeostasis and wherein the animal's health will continue to deteriorate if the disease is not ameliorated.

[0073] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis but the animal's state of health is less favorable than it would be in the absence of the disorder. If untreated, the disorder will not necessarily result in a further decline in the animal's state of health.

[0074] A disease or condition is "reduced" if the signs or symptoms of the disease or condition are reduced in severity, the frequency with which the patient experiences such signs or symptoms, or both.

[0075] "Encoding" refers to the intrinsic properties and resulting biological properties of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA or mRNA) that serves as a template for synthesizing other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process. Therefore, a gene encodes a protein if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a transcription template for a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0076] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or cell that can be applied to the methods described herein, whether in vitro or in vivo. In one embodiment, the subject includes 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, crab-eating monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include, but are not limited to, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chicks, emus, ostriches), and fish (e.g., zebrafish, trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0077] The term "specifically binds" as used herein with respect to antibodies refers to antibodies that recognize a specific antigen but do not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to the antigen from one or more species. However, this species cross-reactivity itself does not change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody.

[0078] In some cases, the term "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical substance to refer to the interaction being dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than binding to proteins broadly. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0079] The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of an mRNA molecule produced by transcription of the gene.

[0080] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that match the anticodon region of the transfer RNA molecule or encode a stop codon during translation of the mRNA molecule. Thus, the coding region may include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0081] As used herein with respect to nucleic acid, "complementarity" refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that the adenine residues of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with the residues of the second nucleic acid region, and if the residues of the second nucleic acid region are thymine or uracil, the second nucleic acid region is antiparallel to the first region. Similarly, it is known that the cytosine residues of the first nucleic acid chain can base pair with the residues of the second nucleic acid chain, and if the residues of the second nucleic acid chain are guanine, the second nucleic acid chain is antiparallel to the first chain. If at least one nucleotide residue in the first region can base pair with the residues in the second region when the two regions are arranged in an antiparallel manner, the first region of the nucleic acid is complementary to the second region of the same or different nucleic acids. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first portion and the second portion are arranged in an antiparallel manner, 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 can base pair with the nucleotide residues in the second portion. In one embodiment, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0082] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0083] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0084] The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated in the production of, for example, antisense molecules, siRNA, ribozymes, etc. An expression vector may contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a specific host organism. In addition to control sequences for transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0085] The term "wild type" as used herein is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene, or characteristic that distinguishes it from mutant or variant forms as it occurs in nature.

[0086] The term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705). This software matches similar sequences by assigning values ​​to the degree of homology of different 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.

[0087] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in its normal environment in a living organism is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment (e.g., a host cell).

[0088] When used in relation to nucleic acids, the term "isolated" 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 normally associated in its source. Thus, isolated nucleic acids exist in a form or environment different from that found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state in which they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome close to adjacent genes; an RNA sequence (e.g., a specific mRNA sequence encoding a specific protein) is found in a cell as a mixture with many other mRNAs encoding a variety of proteins. However, for example, an isolated nucleic acid includes such a nucleic acid in a cell that normally expresses the nucleic acid, wherein the nucleic acid is in a chromosomal position different from that of a natural cell, or is otherwise flanked by a different nucleic acid sequence found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide contains at least the sense or coding strand (ie, the oligonucleotide may be single-stranded), but may contain both the sense and antisense strands (ie, the oligonucleotide may be double-stranded).

[0089] The term "isolated" when used in reference to a polypeptide, as in "isolated protein" or "isolated polypeptide," refers to a polypeptide that has been identified and separated from at least one contaminant with which it is normally associated in its source. Thus, an isolated polypeptide exists in a form or environment different from that found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state in which they exist in nature.

[0090] "Nucleic acid" refers to any nucleic acid, whether it is composed of deoxyribonucleosides or ribonucleosides, and whether it is composed of phosphodiester linkages or modified linkages, such as phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamide esters, carbamates, thioethers, bridged phosphoramidates, bridged methylenephosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, bridged phosphorothioates or sulfone linkages and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases that are different from five biologically existing bases (adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0091] Conventional notation is used herein to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5'-end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.

[0092] The direction in which nucleotides are added 5' to 3' to the nascent RNA transcript is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the "coding strand"; the sequence on the DNA strand that is 5' to a reference point on the DNA is called the "upstream sequence"; the sequence on the DNA strand that is 3' to a reference point on the DNA is called the "downstream sequence".

[0093] "Expression cassette" refers to a nucleic acid molecule comprising a coding sequence operably linked to promoter / regulatory sequences necessary for the transcription and, optionally, translation of the coding sequence.

[0094] As used herein, the term "operably linked" refers to the linkage of nucleic acid sequences in such a way that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a subsequent protein molecule is produced. The term also refers to the linkage of amino acid encoding sequences in such a way that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibition, etc.) protein or polypeptide is produced.

[0095] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for expressing a gene product that is operably linked to a promoter / regulatory factor sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for expressing a gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses a gene product in an inducible manner.

[0096] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence hybridizes primarily with the target sequence and does not substantially hybridize with non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with 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, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0097] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence-specific manner. The complex may comprise two chains forming a duplex structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of PCR or the cleavage of polynucleotides by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the "complementary sequence" of a given sequence.

[0098] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced substantially only in the presence of an inducer corresponding to the promoter.

[0099] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that 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.

[0100] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include but are not limited to all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using common cloning techniques and PCR, etc.) and by synthetic means.

[0101] In the context of the present invention, the following abbreviations for ubiquitous 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.

[0102] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and there is no limit on the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the terms refer to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0103] The term "RNA" as used herein is defined as ribonucleic acid.

[0104] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally linked together. The amplified or assembled recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform a suitable host cell.

[0105] A recombinant polynucleotide may also serve a non-coding function (eg, promoter, origin of replication, ribosome binding site, etc.).

[0106] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by the use of recombinant DNA methodologies.

[0107] As used herein, "variant" is a nucleic acid sequence or peptide sequence whose sequence is different from a reference nucleic acid sequence or peptide sequence, but retains the basic biological properties of the reference molecule. The changes in the sequence of nucleic acid variants may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may produce amino acid substitutions, additions, deletions, fusions and truncations. The changes in the sequence of peptide variants are usually limited or conservative, so that the sequences of the reference polypeptide and variant are generally closely similar and identical in many regions. Variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The variant of nucleic acid or peptide may be naturally occurring, such as an allelic variant, or may be a variant not known to be naturally occurring. The non-naturally occurring variants of nucleic acids and peptides may be prepared by mutagenesis techniques or by direct synthesis.

[0108] "Vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphipathic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0109] Range: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the description in range form is merely for convenience and brevity and should not be interpreted as a hard limit to the scope of the invention. Therefore, the description of a range should be considered to have all possible sub-ranges disclosed as well as individual numerical values ​​within the range. For example, a description of a range such as 1 to 6 should be considered to have sub-ranges disclosed as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies to any width range.

[0110] Fusion Protein

[0111] In one aspect, the present invention is based on the development of novel fusions of editing proteins that are effectively delivered to the nucleus. In one embodiment, the fusion protein is effectively delivered to the nucleus and can cleave nuclear RNA. In one embodiment, the fusion protein is effectively delivered to the nucleus and can visualize nuclear RNA. In one embodiment, the fusion protein is effectively delivered to the nucleus and can regulate the cleavage and / or polyadenylation of nuclear RNA.

[0112] EraseR

[0113] In one aspect, the present invention is based on the development of novel fusions of editing proteins that are effectively delivered to the nucleus. In one aspect, the present invention provides a fusion protein comprising an editing protein having a first amino acid sequence and a nuclear localization signal (NLS) having a second amino acid sequence.

[0114] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0115] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0116] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b,dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, dUrCas13d or variants thereof. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0117] In one embodiment, the Cas protein comprises a sequence that is 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 that is 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 one of SEQ ID NOs: 1-46.

[0118] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleolar phosphoprotein (NPM1) or simian vims 40 ("SV40") T antigen.

[0119] In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77. In one embodiment, the NLS comprises a sequence that is 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 the sequence of one of SEQ ID NOs: 75-695.

[0120] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 the sequence of one of SEQ ID NOs: 83-695.

[0121] 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.

[0122] In one embodiment, the fusion protein comprises a sequence that is 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. In one embodiment, the fusion protein comprises the sequence of SEQ ID NO: 702.

[0123] HilightR

[0124] In one aspect, the present invention is based on the development of novel fusions of editing proteins and fluorescent proteins that are effectively delivered to the nucleus. These fusion proteins combine the visualization ability of fluorescent proteins and the programmable DNA targeting ability of catalytic death Cas. In one embodiment, the invention provides fusion proteins, the fusion proteins comprising CRISPR-related (Cas) proteins with a first amino acid sequence and fluorescent proteins with a second amino acid sequence. In one embodiment, the fusion protein comprises a nuclear localization signal with a third amino acid sequence. In one embodiment, the fusion protein comprises a joint with a fourth amino acid sequence. In one embodiment, the joint connects Cas proteins and fluorescent proteins. In one embodiment, the fusion protein comprises a purification and / or detection label with a fifth amino acid sequence.

[0125] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0126] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0127] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, 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, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0128] In one embodiment, the Cas protein comprises a sequence that is 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 that is 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 with one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions render the Cas protein catalytically inactive. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 1-48. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 47-48.

[0129] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77. In one embodiment, the NLS comprises a sequence that is 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 the sequence of one of SEQ ID NOs: 75-695.

[0130] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 the sequence of one of SEQ ID NOs: 83-695.

[0131] 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.

[0132] In one embodiment, the fluorescent protein is eGFP, mCherry, mCherry-MBNL1, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), 7xS11, sfCherry, S11, Emerald, Superfold GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, blue fluorescent protein, EBFP, EBFP2, Azurite, mTagBFP, cyan fluorescent protein, eCFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1(Teal), yellow fluorescent protein, EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, orange fluorescent protein, Kusabira Orange, KusabiraOrange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-monomer, mTangerine, red fluorescent protein, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0133] In one embodiment, the fluorescent protein is eGFP, mCherry, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry or 7xS11. In one embodiment, the fluorescent protein comprises an amino acid sequence that is 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. In one embodiment, the fluorescent protein comprises the amino acid sequence of one of SEQ ID NOs: 59-66.

[0134] In one embodiment, the fusion protein comprises a purification and / or detection tag. In one embodiment, the tag is located at the N-terminus of the fusion protein. In one embodiment, the tag comprises an amino acid sequence that is 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 the amino acid sequence of SEQ ID NO: 7474.

[0135] In one embodiment, the fusion protein comprises a joint. In one embodiment, the joint connects the Cas protein and the fluorescent protein. In one embodiment, the joint is connected to the C-terminus of the Cas protein and the N-terminus of the fluorescent protein. In one embodiment, the joint comprises 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 sequence to one of SEQ ID NO:67-73. In one embodiment, the joint comprises the sequence of one of SEQ ID NO:67-73.

[0136] In one embodiment, the fusion protein comprises an amino acid sequence that is 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. In one embodiment, the fusion protein comprises an amino acid sequence of one of SEQ ID NOs: 699-701.

[0137] Postscript

[0138] In one aspect, the present invention is based on the development of novel fusions of editing proteins and cleavage and / or polyadenylated proteins effectively delivered to the nucleus. These fusion proteins combine the catalytic activity of cleavage and / or polyadenylated proteins and the programmable DNA targeting ability of catalytic death Cas. In one embodiment, the present invention provides a fusion protein comprising a CRISPR-related (Cas) protein with a first amino acid sequence and a cleavage and / or polyadenylated protein with a second amino acid sequence. In one embodiment, the fusion protein comprises a nuclear localization signal with a third amino acid sequence. In one embodiment, the fusion protein comprises a joint with a fourth amino acid sequence. In one embodiment, a joint connects Cas proteins and cleavage and / or polyadenylated proteins. In one embodiment, the fusion protein comprises a purification and / or detection label with a fifth amino acid sequence.

[0139] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0140] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0141] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, 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, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0142] In one embodiment, the Cas protein comprises a sequence that is 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 that is 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 with one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions render the Cas protein catalytically inactive. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 1-48. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 47-48.

[0143] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77. In one embodiment, the NLS comprises a sequence that is 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 the sequence of one of SEQ ID NOs: 75-695.

[0144] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 the sequence of one of SEQ ID NOs: 83-695.

[0145] 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.

[0146] In one embodiment, the cleavage and / or polyadenylation protein is an RNA binding protein of the human 3' end processing machinery. In one embodiment, the cleavage and / or polyadenylation protein is CPSF30, WDR33 or NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21, a NUDT21 mutation, a NUDT21 dimer, a NUDT21 fusion protein or any combination thereof. In one embodiment, the cleavage and / or polyadenylation protein is a tandem dimer of human NUDT21, worm NUDT21, fly NUDT21, zebrafish NUDT21NUDT21_R63S, NUDT21_F103A or NUDT21.

[0147] In one embodiment, the cleavage and / or polyadenylation protein comprises an amino acid sequence that is 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 that is 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 and / or polyadenylation protein comprises the amino acid sequence of SEQ ID NOs: 51-58.

[0148] In one embodiment, the fusion protein comprises a purification and / or detection tag. In one embodiment, the tag is located at the N-terminus of the fusion protein. In one embodiment, the tag comprises an amino acid sequence that is 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 the amino acid sequence of SEQ ID NO:74.

[0149] In one embodiment, the fusion protein comprises a joint. In one embodiment, the joint connects the Cas protein and the cleavage and / or polyadenylation protein. In one embodiment, the joint is connected to the C-terminus of the Cas protein and the N-terminus of the cleavage and / or polyadenylation protein. In one embodiment, the joint comprises a sequence that is 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 NO:67-73. In one embodiment, the joint comprises a sequence of one of SEQ ID NO:67-73.

[0150] In one embodiment, the fusion protein comprises an amino acid sequence that is 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.

[0151] The fusion protein of the present invention can be prepared using chemical methods. For example, the fusion protein can be synthesized by solid phase technology (Roberge JY et al. (1995) Science 269: 202-204), cleaved from resin, and purified by preparative high performance liquid chromatography. Automatic synthesis can be achieved, for example, using ABI 431 A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0152] The present invention should also be interpreted as including any form of fusion proteins having substantial homology to the fusion proteins disclosed herein. In one embodiment, a "substantially homologous" fusion protein is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous, about 95% homologous, or about 99% homologous to the amino acid sequence of the fusion protein disclosed herein.

[0153] Fusion proteins can alternatively be prepared by recombinant means or by cleavage from longer polypeptides. The composition of the fusion protein can be confirmed by amino acid analysis or sequencing.

[0154] Variants of fusion proteins according to the present invention may be: (i) fusion proteins in which one or more of the amino acid residues are substituted with conservative or non-conservative amino acid residues and such substituted amino acid residues may or may not be residues encoded by the genetic code, (ii) fusion proteins in which there are one or more modified amino acid residues (e.g., residues modified by connecting substituents), (iii) fusion proteins in which the peptide is an alternative splicing variant of the fusion protein of the present invention, (iv) fragments of the peptide and / or (v) fusion proteins in which the fusion protein is fused to another peptide (such as a leader sequence or secretory sequence or a sequence used for purification (e.g., His-tag) or for detection (e.g., Sv5 epitope tag). The fragments include peptides generated by proteolytic cleavage of the original sequence (including multisite proteolysis). Variants may be post-translationally modified or chemically modified. Such variants should be within the scope of those skilled in the art according to the teachings herein.

[0155] As known in the art, the "similarity" between two fusion proteins is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitute with the sequence of the second polypeptide. Variants are defined as peptide sequences that include different from the original sequence. In one embodiment, the variant is different from the original sequence in less than 40% of the residues in each target segment, different from the original sequence in less than 25% of the residues in each target segment, different from the original sequence in less than 10% of the residues in each target segment, or different from the original protein sequence in only some residues in each target segment, and is sufficiently homologous to the original sequence at the same time, to retain the functionality of the original sequence and / or stimulate stem cells to differentiate into the ability of 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. Computer algorithms and methods widely known to those skilled in the art are used to determine the degree of identity between two peptides. The identity between two amino acid sequences can 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)].

[0156] Fusion protein of the present invention can be post-translationally modified. For example, post-translation modifications falling 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 need to introduce additional biological machinery. For example, processing events (such as signal peptide cleavage and core glycosylation) are tested by adding canine microsomal membranes or African clawed frog egg extracts (U.S. Patent number 6,103,489) to standard translation reactions.

[0157] The fusion proteins of the present invention may comprise unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. Various methods are available for introducing unnatural amino acids during protein translation.

[0158] The fusion proteins of the present invention can be phosphorylated using conventional methods, such as the method described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0159] Cyclic derivatives of the fusion protein of the present invention are also part of the present invention. Cyclization can make the fusion protein present a more favorable conformation for association with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between the amino group of one component and the carboxyl group of another component. Cyclization can also be achieved using azobenzene-containing amino acids, as described by Ulysse, L., et al., J.Am.Chem.Soc.1995, 117, 8466-8467. The components forming the bond can be side chains of amino acids, non-amino acid components, or a combination of the two. In one embodiment of the present invention, the cyclic peptide may be included in a β-turn in the right position. The β-turn can be introduced into the peptide of the present invention by adding the amino acid Pro-Gly at the right position.

[0160] It may be desirable to produce a more flexible cyclic fusion protein than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to deform the β-sheet and the β-turn. Due to the length of the disulfide bonds in the β-sheet portion and the smaller number of hydrogen bonds, the peptide is more flexible. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulations.

[0161] The present invention also relates to peptides comprising fusion proteins comprising Cas13 and a fluorescent protein or Cas13 and a cleavage and / or polyadenylation protein, wherein the fusion protein itself is fused to or integrated into a target protein and / or a targeting domain that can direct the chimeric protein to a desired cellular component or cell type or tissue. The chimeric protein may also contain additional amino acid sequences or domains. The chimeric protein is recombinant because the various components come from different sources and because such components do not exist together in nature (i.e., are heterologous).

[0162] In one embodiment, the targeting domain can be a transmembrane domain, a membrane binding domain, or a sequence that directs the protein to associate, for example, with a vesicle or with a nucleus. In one embodiment, the targeting domain can target the peptide to a specific cell type or tissue. For example, the targeting domain can be a cell surface ligand or antibody to a cell surface antigen of the target tissue. The targeting domain can target the peptides of the invention to cellular components.

[0163] The peptide of the present invention can be synthesized by conventional techniques. For example, the peptide or chimeric protein can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods adopt solid phase or liquid phase synthesis methods (see, for example, about solid phase synthesis technology JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RBM Merrifield, The Peptides: Analysis Synthesis, Biology edited E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; and about classical solution synthesis M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, editors, The Peptides: Analysis, Synthesis, Biology, suprs, Vol. 1). By way of example, the peptides of the invention can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry, wherein phosphothreonine is directly incorporated as an N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0164] The N-terminal or C-terminal fusion protein comprising a peptide or chimeric protein of the present invention conjugated to other molecules can be prepared by recombinant technology by fusing the N-terminal or C-terminal of the peptide or chimeric protein with a sequence of a selected protein or a selective marker having a desired biological function. The resulting fusion protein contains a fusion protein fused to a selected protein or marker protein as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0165] The peptides of the present invention can be developed using biological expression systems. Using these systems allows large libraries of random peptide sequences to be generated and these libraries to be screened for peptide sequences that bind to specific proteins. Libraries can be generated by cloning synthetic DNA encoding 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 can also be constructed by parallel synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0166] The peptides and chimeric proteins of the present invention can 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, benzenesulfonic acid and toluenesulfonic acid.

[0167] Nucleic Acids

[0168] In one aspect, the present invention is based on the development of nucleic acids encoding novel fusions of editing proteins that 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 can cleave nuclear RNA. In one embodiment, the nucleic acid encodes a fusion protein that can be effectively delivered to the nucleus and can visualize nuclear RNA. In one embodiment, the nucleic acid encodes a fusion protein that can be effectively delivered to the nucleus and can regulate the cleavage and / or polyadenylation of nuclear RNA.

[0169] EraseR

[0170] In one embodiment, the present invention provides a nucleic acid molecule encoding a fusion protein. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an editing protein; and a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0171] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0172] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0173] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, 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, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0174] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-46.

[0175] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is 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 the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 707-708.

[0176] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleolar phosphatidylcholine (NPM1) or simian vims 40 ("SV40") T antigen.

[0177] In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77.

[0178] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 NLS comprises the 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 the sequence of one of SEQ ID NOs: 83-695.

[0179] 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.

[0180] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is 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 the NLS comprises the nucleic acid sequence of SEQ ID NO: 724.

[0181] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence encoding an amino acid sequence that is 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. In one embodiment, the fusion protein comprises the sequence of SEQ ID NO: 702. In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence encoding the amino acid sequence of SEQ ID NO: 702.

[0182] HilightR

[0183] In one aspect, the present invention is based on the development of novel nucleic acids encoding fusion proteins, and the fusion proteins include editing proteins and fluorescent proteins that are effectively delivered to the nucleus. In one embodiment, the nucleic acid molecule includes a nucleic acid sequence encoding an editing protein; and a nucleic acid sequence encoding a fluorescent protein. In one embodiment, the nucleic acid molecule includes a nucleic acid sequence encoding a nuclear localization signal (NLS). In one embodiment, the nucleic acid molecule includes a nucleic acid sequence encoding a joint. In one embodiment, the nucleic acid molecule includes a nucleic acid sequence encoding a tag.

[0184] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0185] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0186] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, 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, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0187] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the 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 the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-46 with one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions render the Cas protein catalytically inactive. The nucleic acid sequence encoding the 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 the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 47-48.

[0188] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is 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 the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 707-708.

[0189] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleolar phosphatidylcholine (NPM1) or simian vims 40 ("SV40") T antigen.

[0190] In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77.

[0191] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 the sequence of one of SEQ ID NOs: 83-695.

[0192] 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.

[0193] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is 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 the NLS comprises the nucleic acid sequence of SEQ ID NO: 724.

[0194] In one embodiment, the fluorescent protein is eGFP, mCherry, mCherry-MB NL1, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry 7xS11, S11, Emerald, Superfold GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, blue fluorescent protein, EBFP, EBFP2, Azurite, mTagBFP, cyan fluorescent protein, eCFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1(Teal), yellow fluorescent protein, EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, orange fluorescent protein, Kusabira Orange, KusabiraOrange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-monomer, mTangerine, red fluorescent protein, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0195] In one embodiment, the fluorescent protein is eGFP, mCherry, sfGFP, sfGFP(1-10), sfGFP(1-10)-L-(11), sfCherry or 7xS11. In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence that is 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. In one embodiment, the nucleic acid molecule comprises a sequence encoding the amino acid sequence of one of SEQ ID NOs: 59-66.

[0196] In one embodiment, the nucleic acid molecule comprises a sequence that is 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. In one embodiment, the nucleic acid molecule comprises the sequence of one of SEQ ID NOs: 718-721.

[0197] 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 that is 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 the amino acid sequence of SEQ ID NO: 7474.

[0198] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises the sequence of SEQ ID NO: 723.

[0199] 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 that is 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: 67-73.74.

[0200] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises the sequence of SEQ ID NO: 722723.

[0201] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence encoding an amino acid sequence that is 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. In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 699-701.

[0202] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence that is 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. In one embodiment, the nucleic acid molecule encoding the fusion protein comprises the sequence of one of SEQ ID NOs: 728-730.

[0203] PostscriptR

[0204] In one aspect, the present invention is based on the development of novel nucleic acids encoding fusion proteins, the fusion proteins comprising editing proteins and cleavage and / or polyadenylation proteins effectively delivered to the nucleus. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding the editing protein; and a nucleic acid sequence encoding cleavage and / or polyadenylation. 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 joint. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a tag.

[0205] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0206] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, 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, AsCpf1, LbCpf1, FnCpf1, 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 the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave 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 the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0207] In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas 13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas1 3b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13 a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c,d FnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dL spCas13a, 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, 173:665-676e14; Yan et al., Mol Cell, 2018, 7:327-339e5; Cox, DBT, 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, 66:373-383e3, which are incorporated herein by reference).

[0208] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the 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 the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-46 with one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions render the Cas protein catalytically inactive. The nucleic acid sequence encoding the 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 the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 47-48.

[0209] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is 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 the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 707-708.

[0210] 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 protein, 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 virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleolar phosphatidylcholine (NPM1) or simian vims 40 ("SV40") T antigen.

[0211] In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 75. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 76. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 77.

[0212] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. 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 that is 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 the 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 the sequence of one of SEQ ID NOs: 83-695.

[0213] 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.

[0214] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is 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 the NLS comprises the nucleic acid sequence of SEQ ID NO: 724.

[0215] In one embodiment, the cleavage and / or polyadenylation protein is an RNA binding protein of the human 3' end processing machinery. In one embodiment, the cleavage and / or polyadenylation protein is CPSF30, WDR33 or NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21. In one embodiment, the cleavage and / or polyadenylation protein is NUDT21, a NUDT21 mutation, a NUDT21 dimer, a NUDT21 fusion protein or any combination thereof. 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.

[0216] In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence that is 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 nucleic acid molecule comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 49-58.

[0217] In one embodiment, the nucleic acid molecule comprises a sequence encoding an amino acid sequence that is 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 nucleic acid molecule comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 51-58.

[0218] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises a sequence of one of SEQ ID NOs: 711709-717.

[0219] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises the sequence of one of SEQ ID NOs: 709-717.

[0220] 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 that is 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 the amino acid sequence of SEQ ID NO: 7474.

[0221] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises the sequence of SEQ ID NO: 723.

[0222] 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 that is 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: 67-73.74.

[0223] In one embodiment, the nucleic acid molecule comprises a sequence that is 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, the nucleic acid molecule comprises the sequence of SEQ ID NO: 722723.

[0224] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence encoding an amino acid sequence that is 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 the fusion protein comprises a sequence encoding an amino acid sequence of one of SEQ ID NOs: 696-698.

[0225] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence that is 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 the fusion protein comprises the sequence of one of SEQ ID NOs: 725-727.

[0226] The isolated nucleic acid sequence encoding the fusion protein can be obtained using any of the many recombinant methods known in the art, such as by screening a library from cells expressing the gene, by obtaining the gene from a vector known to contain the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the target gene can be produced synthetically rather than cloned.

[0227] The nucleic acid of separation can comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises the DNA molecule of separation, including, for example, the cDNA molecule of separation encoding the fusion protein of the present invention. In one embodiment, the composition comprises the RNA molecule of separation encoding the fusion protein of the present invention or its functional fragment.

[0228] Nucleic acid molecules of the present invention can be modified to improve stability in serum or in a growth medium for cell culture. Modifications can be added to enhance the stability, functionality and / or specificity of nucleic acid molecules of the present invention and to minimize immunostimulatory properties. For example, in order to enhance stability, 3'-residues can be stabilized for degradation, for example, they can be selected so that they consist of purine nucleotides, specifically adenosine or guanosine nucleotides. Alternatively, replacing pyrimidine nucleotides with modified analogs, for example replacing uridine with 2'-deoxythymidine is tolerable and does not affect the function of the molecule.

[0229] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0230] The limiting examples of nucleotide analogs include sugar-modified and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one of nitrogen or sulfur heteroatoms. In exemplary backbone-modified ribonucleotides, the phosphate groups connected to adjacent ribonucleotides are replaced by modified groups (e.g., modified groups of thiophosphate groups). In exemplary sugar-modified ribonucleotides, 2'OH-groups are replaced by groups selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0231] Other examples of modifications are ribonucleotides modified with nucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The 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, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8 position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications may be combined.

[0232] In some cases, nucleic acid molecules include at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modifications of one or more nucleotides. In certain embodiments, nucleic acid molecules of the present invention may have enhanced resistance to nucleases. For increased nuclease resistance, nucleic acid molecules may include, for example, 2'-modified ribose units and / or thiophosphate linkages. For example, 2'hydroxyl (OH) can be modified or replaced using a variety of different "oxy" or "deoxy" substituents. For enhanced nuclease resistance, nucleic acid molecules of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate linkages. Comprising locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid) and certain core base modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications) may also increase binding affinity to the target.

[0233] In one embodiment, the nucleic acid molecule comprises 2'-modified nucleotides, such as 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'-ON-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0234] In certain embodiments, nucleic acid molecules of the invention have one or more of the following properties:

[0235] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA and polymers modified, for example, to improve efficacy RNA and DNA and nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of nucleic acid (i.e., sugar, base and phosphate moiety) are identical or substantially identical to the components present in nature or to the components naturally present in the human body. Rare or unusual but naturally occurring RNA is referred to as modified RNA in the art, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or unusual RNA (often referred to as modified RNA) is usually the result of post-transcriptional modification and is within the term unmodified RNA as used herein. Modified RNA as used herein refers to one or more of the components of nucleic acid (i.e., sugar, base and phosphate moiety) that are different from the components present in nature, or different from the components present in the human body. Although they are referred to as "modified RNA", because of modification, they certainly include molecules that are not strictly RNA. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct (e.g., an uncharged mimetic of the ribose phosphate backbone) that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with the ribose phosphate backbone.

[0236] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobases.

[0237] The present invention also includes a vector into which the isolated nucleic acid of the present invention is inserted. There are many suitable vectors that can be used in the present invention in the art.

[0238] In short, the expression of the natural or synthetic nucleic acid encoding the fusion protein of the present invention is usually achieved by operably connecting the nucleic acid encoding the fusion protein of the present invention or its part to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication in eukaryotic cells and is optionally integrated into eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0239] The vectors of the present invention can 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, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0240] The isolated nucleic acid of the present invention can be cloned into various 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. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0241] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, and is described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site and one or more selective markers (such as WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193) that function in at least one organism.

[0242] Delivery systems and methods

[0243] In one aspect, the present invention relates to the development of novel lentiviral packaging and delivery systems. Lentiviral particles deliver viral enzymes as proteins. In this way, the lifespan of lentiviral enzymes is short, thereby limiting the possibility of off-target editing due to long-term expression throughout the entire life cycle of the cell. Considering that their required activity is only required for a short period of time, it is advantageous to incorporate editing components or traditional CRISPR-Cas editing components as proteins into lentiviral particles. Therefore, in one embodiment, the present invention provides a lentiviral delivery system, as well as a method for delivering a composition of the present invention, editing genetic material, and using a lentiviral delivery system for nucleic acid delivery.

[0244] 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 a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a D116N integrase mutation.

[0245] 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.

[0246] In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, Cas protein and one or more of NLS, cleavage and / or polyadenylation protein and fluorescent protein. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, Cas protein and one or more of NLS, cleavage and / or polyadenylation protein and fluorescent protein. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, Cas protein and one or more of NLS, cleavage and / or polyadenylation protein and fluorescent protein. In one embodiment, the fusion protein comprises a protease cleavage site between VPR and Cas protein NLS, cleavage and / or polyadenylation protein or fluorescent protein. In one embodiment, the VPR plasmid packaging plasmid further comprises a sequence encoding a guide RNA sequence.

[0247] In one embodiment, a packaging plasmid, a transfer plasmid, an envelope plasmid, and a VPR plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes and translates a fusion protein to produce a VPR fusion protein. In one embodiment, the cell transcribes and translates a fusion protein to produce a VPR fusion protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding a guide RNA.

[0248] In one embodiment, the gag-pol protein, the envelope polyprotein, and the VPR-fusion protein bound to the guide RNA are packaged into viral particles. In one embodiment, the viral particles are collected from the cell culture medium. In one embodiment, the VPR is cleaved from the fusion protein in the viral particles via a protease site to provide a Cas-fusion protein. In one embodiment, the viral particles transduce target cells, wherein the guide RNA binds to the target region of the RNA, thereby targeting the Cas fusion protein.

[0249] In addition, a variety of other virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The 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 the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0250] For example, vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its progeny in daughter cells. Lentiviruses have the added advantage over those derived from oncogenic retroviruses (such as murine leukemia viruses) because lentiviruses can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0251] In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). The term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, and the adeno-associated virus serotype includes but is not limited to 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 treating various diseases. AAV vectors have a variety of features that make them ideally suitable for gene therapy, including lack of pathogenicity, minimum immunogenicity and the ability to transduce post-mitotic cells in a stable and efficient manner. The expression of the specific gene contained in the AAV vector can be specifically targeted to one or more types of cells by selecting an appropriate combination of AAV serotype, promoter and delivery method.

[0252] AAV vectors may have one or more of the AAV wild-type genes, preferably the rep and / or cap genes, completely or partially deleted, but retain functional flanking ITR sequences. Despite their high homology, different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, it is known that AAV1 transduces skeletal and cardiac muscle more efficiently than AAV2. Since most studies have been done with pseudotyped vectors, in which vector DNA flanked by AAV2 ITRs is packaged into capsids of alternative serotypes, it is clear that the biological differences are related to the capsid rather than the genome. Recent evidence suggests that DNA expression cassettes packaged in AAV 1 capsids are at least 1 log 10 more efficient at transducing cardiomyocytes than DNA expression cassettes 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 (AAV1), 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).

[0253] The desired AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3 and hypervariable regions; rep proteins, including rep 78, rep 68, rep 52 and rep 40; and sequences encoding these proteins. These fragments can be easily used in a variety of vector systems and host cells. Such fragments can 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, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be produced by any suitable technique using a combination of selected AAV sequences (e.g., fragments of vp1 capsid proteins) and heterologous sequences, which can be obtained from different selected AAV serotypes, non-continuous parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Thus, exemplary AAVs or artificial AAVs suitable for expressing 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.

[0254] In certain embodiments, the vector further comprises conventional control elements, which are operably connected to the transgene in a manner that allows it to be transcribed, translated and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, "operably connected" sequences include expression control sequences adjacent to the target gene and expression control sequences that act in trans or at a distance to control the target gene. Expression control sequences include: appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product when necessary. A variety of expression control sequences (including natural, constitutive, inducible and / or tissue-specific promoters) are known in the art and can be utilized.

[0255] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these promoters are located in the 30-110bp region upstream of the start site, but many promoters have recently been shown to also contain functional elements downstream of the start site. The intervals between promoter elements are often flexible, so that promoter function is retained when elements are reversed or moved relative to each other. In the thymidine kinase (tk) promoter, before the activity begins to decline, the intervals between promoter elements can be increased to 50bp apart. Depending on the promoter, it seems that individual elements can act collaboratively or independently to activate transcription.

[0256] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a powerful constitutive promoter sequence that is capable of driving high-level expression of any polynucleotide sequence operably 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 simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous (Rous) sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters may also be considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to it when such expression is desired, or turn off such expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0257] The enhancer sequence present on the vector also regulates the expression of the gene contained therein. Generally, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific to enhance the transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention contains one or more enhancers to strengthen the transcription of the gene present in the vector.

[0258] In order to evaluate the expression of fusion protein of the present invention, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both, so as to help identify and select expressing cells from a cell population attempted to be transfected or infected by a viral vector. In other aspects, the selective marker may be carried on a separate DNA and used in a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by appropriate regulator sequences to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo, etc.

[0259] Reporter gene is used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter gene is a gene that is not present in a recipient organism or tissue or is not expressed by the recipient organism or tissue and encodes a polypeptide that is expressed by some properties (e.g., enzymatic activity) that are easy to detect. At the appropriate time after DNA has been introduced into the recipient cell, the expression of reporter gene is measured. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well known, and known technology can be used to prepare or commercially obtain. In general, the construct with the minimum 5' flanking region of the highest level expression of display reporter gene is identified as promoter. Such promoter region can be connected to reporter gene, and is used to assess the ability of promoter-driven transcription.

[0260] Methods for introducing genes into cells and expressing genes are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells (e.g., mammals, bacteria, yeast or insect cells) by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means.

[0261] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing 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 introducing polynucleotides into host cells is calcium phosphate transfection.

[0262] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0263] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0264] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used for introducing nucleic acids into a host cell (in vitro, in vitro or in vivo). In another aspect, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule associated with both the liposome and the oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids in the form of a suspension, contained in micelles or compounded with micelles, or otherwise associated with lipids. The composition associated with lipids, lipid / DNA or lipid / expression vectors is not limited to any specific structure in the solution. For example, they may be present in a double-layer structure, exist in the form of micelles, or have a "collapsed" structure. They may also be only dispersed in the solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, and they can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0265] Lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dihexadecyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available 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 sole solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a variety of monolayer and multilayer lipid vehicles that are formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure, and water and dissolved solutes are embedded between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having different structures in solution compared with normal vesicle structures are also contemplated. For example, lipids may present micellar structures or may exist only in the form of non-uniform lipid molecule aggregates. Lipofectamine-nucleic acid complexes are also contemplated.

[0266] Regardless of the method used to introduce the exogenous nucleic acid into the host cell, various assays can be performed to confirm that the recombinant DNA sequence is present in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of droplet polypeptides, such as by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of the present invention.

[0267] system

[0268] In one aspect, the present invention provides a system for regulating the cleavage, polyadenylation or both of RNA transcripts. In one embodiment, the system comprises a nucleic acid sequence encoding a fusion protein in one or more vectors, wherein the fusion protein comprises a CRISPR-related (Cas) protein, a cleavage and / or polyadenylation protein, and a nuclear localization signal (NLS); and a nucleic acid sequence encoding a CRISPR-Cas system crRNA. In one embodiment, the CRISPR-Cas system crRNA is substantially hybridized with a target RNA sequence in an RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the 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 encoding the CRISPR-Cas system crRNA are in different vectors.

[0269] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises (1) a nucleic acid sequence encoding an amino acid sequence that is 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 that is 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; NO:51-58 is 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 NO:51-58; and (3) encoding an amino acid sequence that is 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 NO:51-58; NO:75-695 is 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 sequences. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence encoding an amino acid sequence that is 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 the fusion protein comprises (1) a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 1-48; (2) a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 51-58; and (3) a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 696-698.

[0270] In one aspect, the present invention provides a system for reducing the number of RNA transcripts in a subject. In one embodiment, the system comprises a nucleic acid sequence encoding a fusion protein in one or more vectors, wherein the fusion protein comprises a CRISPR-related (Cas) protein and a nuclear localization signal (NLS); and a nucleic acid sequence encoding a CRISPR-Cas system crRNA. In one embodiment, the CRISPR-Cas system crRNA is substantially hybridized with a target RNA sequence in an RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the 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 encoding the CRISPR-Cas system crRNA are in different vectors.

[0271] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises (1) a nucleic acid sequence encoding an amino acid sequence that is 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 that is 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. NO:75-695 is 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 sequences. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence encoding an amino acid that is 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. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises (1) a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 1-48; and (2) a nucleic acid sequence encoding an amino acid of one of SEQ ID NOs: 75-695. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence encoding the amino acid of SEQ ID NO:702.

[0272] In one aspect, the present invention provides a system for visualizing nuclear RNA in a subject. In one embodiment, the system includes a nucleic acid sequence encoding a fusion protein in one or more vectors, wherein the fusion protein includes a CRISPR-related (Cas) protein, a fluorescent protein, and a nuclear localization signal (NLS); and a nucleic acid sequence encoding a CRISPR-Cas system crRNA. In one embodiment, the CRISPR-Cas system crRNA is substantially hybridized with a target RNA sequence in an RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the 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 encoding the CRISPR-Cas system crRNA are in different vectors.

[0273] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises (1) a nucleic acid sequence encoding an amino acid sequence that is 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 that is 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; NO:59-66 is 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 that is 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; NO:699-701. A nucleic acid sequence that is 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 amino acid sequences. A nucleic acid sequence encoding an amino acid sequence that is 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. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises (1) a nucleic acid sequence encoding the amino acids of one of SEQ ID NOs: 1-48; (2) a nucleic acid sequence encoding the amino acids of one of SEQ ID NOs: 59-66; and (3) a nucleic acid sequence encoding the amino acids of one of SEQ ID NOs: 75-695.In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence encoding the amino acid of one of SEQ ID NOs: 699-701.

[0274] Systems and vectors can be designed to express 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 further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and a T7 polymerase.

[0275] The vector can be introduced into a prokaryotic organism and propagated therein. In some embodiments, the prokaryotic organism is used to amplify the vector to be introduced into a eukaryotic cell or as a copy of the intermediate vector in the vector to be introduced into a eukaryotic cell (for example, amplification plasmid as part of a viral vector packaging system). In some embodiments, the prokaryotic organism is used to amplify multiple copies of the vector and express one or more nucleic acids to provide a source of one or more proteins for delivery to a host cell or host organism. The expression of proteins in prokaryotes is most often carried out in Escherichia coli with a vector containing a constitutive or inducible promoter that directs the expression of fusion proteins or non-fusion proteins. The fusion vector adds many amino acids to the protein encoded therein, such as being added to the amino terminus of the recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) helping to purify the recombinant protein by acting as a ligand in affinity purification. Often, in a fusion expression vector, a protein cleavage site is introduced at the junction of the fusion part and the recombinant protein so that the recombinant protein can be separated from the fusion part after the fusion protein is purified. Such enzymes and their homologous recognition sequences include factor Xa, thrombin and enterokinase. Examples of 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, NJ), which fuse glutathione S-transferase (GST), maltose E binding protein or protein A to the target recombinant protein, respectively.

[0276] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amran et al., (1988) Gene 69:301-315) and pET11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0277] In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae 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.).

[0278] In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. Baculovirus vectors that can be used to express 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).

[0279] In some embodiments, a mammalian expression vector is used, and the vector is capable of driving one or more sequences to be expressed in mammalian cells. 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 control function of the expression vector is generally provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters disclosed herein and known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, see, for example, Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 16 and 17.

[0280] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (eg, using tissue-specific regulatory elements 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 for 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., 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., whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546).

[0281] In some embodiments, the regulatory element is operably connected to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. In general, CRISPR (clustered regularly spaced short palindromic repeats), also known as SPIDR (Spacer spaced direct repeats), constitutes a family of DNA loci that are usually specific for a particular bacterial species. The CRISPR locus includes a unique class of interspersed short sequence repeats (SSRs) identified in Escherichia coli (Ishino et al., J. Bacteriol., 169: 5429-5433

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

[1989] ) and related genes. Similar interspersed SSRs have also been identified in Halophilus mediterraneus, 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] ). CRISPR loci are generally different from other SSRs in the structure of the repeat sequences, which are called short regularly interspaced repeats (SRSRs) (Janssen et al., OMICS J. Inte g. Biol., 6:23-33

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

[2000] ). In general, the repeat sequences are short elements that exist in clusters and are regularly spaced apart by unique insert sequences of essentially constant length (Mojica et al.,

[2000] , supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeat sequences and the sequence of the spacers often vary 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, Archaeoglobium, and Pseudomonas. us), 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.

[0282] As used herein, "target sequence" refers to a sequence to which the crRNA sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be in an organelle of a eukaryotic cell, such as a mitochondria or a chloroplast.

[0283] The ability of crRNA to guide the sequence-specific binding of nucleic acid targeting complexes to target nucleic acid sequences can be evaluated by any suitable assay. For example, components of a nucleic acid targeting CRISPR system sufficient to form a nucleic acid targeting complex (including a guide sequence to be tested) can be provided to a host cell having a corresponding target nucleic acid sequence, such as by transfection with a vector encoding a component of the nucleic acid targeting complex, and then the preferential targeting (e.g., cleavage) within the target nucleic acid sequence is evaluated by a Surveyor assay as described herein. Similarly, the cleavage of a target nucleic acid sequence can be evaluated in a test tube by providing a target nucleic acid sequence, components of a nucleic acid targeting complex (including a guide sequence to be tested and a control guide sequence different from the test guide sequence) and comparing the binding or cleavage rate at the target sequence between the test guide sequence and the control guide sequence reaction. Other assays are possible and will occur to those skilled in the art. The crRNA sequence can be selected and the nucleic acid targeting crRNA can therefore be selected to target any target nucleic acid sequence.

[0284] The target sequence can be any RNA sequence. In some embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), precursor mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (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 cytoplasmic RNA (scRNA). In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of: mRNA, precursor-mRNA and rRNA. In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of: ncRNA and lncRNA. In some more preferred embodiments, the target sequence can be a sequence within an mRNA molecule or a precursor mRNA molecule.

[0285] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, which was developed by the Institute of Theoretical Chemistry of the University of Vienna, using a centroid structure prediction algorithm (see, for example, ARGruber et al., 2008, Cell 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

[0286] In some embodiments, crRNA is selected to reduce the degree of secondary structure within the nucleic acid targeting guide. In some embodiments, when optimally folded, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleotides of the nucleic acid targeting guide participate in self-complementary base pairing. The optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, which was developed by the Institute of Theoretical Chemistry at the University of Vienna using the centroid structure prediction algorithm (see, eg, AR Gruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0287] In certain embodiments, crRNA may include, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or a spacer sequence. In certain embodiments, crRNA may include, consist essentially of, or consist of a direct repeat sequence fused or connected to a guide sequence or a spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., 5') of a guide sequence or a spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') of a guide sequence or a spacer sequence.

[0288] In certain embodiments, the crRNA comprises a stem loop. In one embodiment, the crRNA comprises a single stem loop. In certain embodiments, the same direction repeat sequence forms a stem loop. In one embodiment, the same direction repeat sequence forms a single stem loop.

[0289] In one embodiment, crRNA enhances the activity of Cas13 targeting target sequence, Cas13 catalytic activity or both. For example, in one embodiment, the crRNA crRNA sequence comprises a mutation in its direct repeat sequence. For example, in one embodiment, crRNA comprises a T17C point mutation. In one embodiment, crRNA comprises a T18C point mutation.

[0290] Pharmaceutical compositions and preparations

[0291] The present invention also encompasses the use of the pharmaceutical composition of the present invention or its salt for implementing the method of the present invention. Such pharmaceutical compositions may be composed of at least one modulator (e.g., inhibitor or activator) composition of the present invention or its salt in a form suitable for administration to a subject, or the pharmaceutical composition may include at least one modulator (e.g., inhibitor or activator) composition of the present invention or its salt, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compounds of the present invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt combined with a physiologically acceptable cation or anion, as is well known in the art.

[0292] In one embodiment, a pharmaceutical composition suitable for practicing the methods of the present invention may be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, a pharmaceutical composition suitable for practicing the present invention may be administered to deliver a dose between 1 ng / kg / day and 500 mg / kg / day.

[0293] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of the present invention will vary depending on the identity, size, and condition of the subject being treated, and further depend on the route by which the composition is to be administered. For example, the composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0294] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular or another route of administration. Compositions useful within the methods of the present invention may be applied directly to the skin or any other tissue of a mammal. Other contemplated formulations include liposomal formulations, resealed erythrocytes containing the active ingredient, and immunologically based formulations. The route of administration will be apparent to the skilled artisan and will depend on a number of factors, including the type and severity of the disease being treated, the type and age of the livestock or human subject being treated, and the like.

[0295] The preparations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the steps of combining the active ingredient with a carrier or one or more other auxiliary ingredients and then, if necessary or desirable, forming or packaging the product into a desired single or multiple dosage unit.

[0296] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to the subject or a convenient fraction of such a dose, such as one-half or one-third of such a dose. 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.

[0297] In one embodiment, the composition of the invention is formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical composition of the invention comprises a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Available pharmaceutically acceptable carriers 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).

[0298] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The effect of preventing microorganisms can be achieved by a variety of antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, for example, sugar, sodium chloride or polyols such as mannitol and sorbitol are included in the composition. The absorption extension of the injectable composition can be achieved by including an agent such as aluminum monostearate or gelatin that delays absorption in the composition. In one embodiment, the pharmaceutically acceptable carrier is not only DMSO.

[0299] The preparation can be used in combination with conventional excipients suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable mode of administration known in the art, i.e., pharmaceutically acceptable organic or inorganic carrier materials. The pharmaceutical preparation can be sterilized and mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, colorants, flavorings and / or aromatic substances, etc. when necessary. It can also be combined with other active agents (e.g., other analgesics) when necessary.

[0300] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavoring agents; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, sedatives; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0301] The compositions of the present invention may contain about 0.005% to 2.0% preservatives by total weight of the composition. Preservatives are used to prevent spoilage in the event of exposure to pollutants in the environment. Examples of preservatives that can be used according to the present invention include, but are not limited to, those preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidureas, 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.

[0302] In one embodiment, the composition comprises an antioxidant and a chelating agent that inhibits the degradation of the compound. Exemplary antioxidants of some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid at about 0.01% to 0.3% by weight of the total weight of the composition, and BHT is in the range of 0.03% to 0.1% by weight. In one embodiment, by the total weight of the composition, the chelating agent is present in an amount of 0.01% to 0.5% by weight. Exemplary chelating agents include edetates (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%. In some embodiments, by the total weight of the composition, the chelating agent is in the range of 0.02% to 0.10% by weight. Chelating agents can be used to chelate metal ions in the composition that may be harmful to the shelf life of the preparation. Although BHT and disodium edetate are exemplary antioxidants and chelating agents of some compounds, respectively, other suitable and equivalent antioxidants and chelating agents can be substituted, as known to those skilled in the art.

[0303] Conventional methods can be used to prepare liquid suspensions to achieve suspension of active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethanol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin. Liquid suspensions may also include one or more additional ingredients, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, sedatives, preservatives, buffers, salts, flavoring agents, coloring agents and sweeteners. Oily suspensions may also include thickening agents. Known suspending agents include but are not limited to sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersants or wetting agents include but are not limited to naturally occurring phosphatides (such as lecithin), alkylene oxides and fatty acids, long-chain fatty alcohols, partial esters derived from fatty acids and hexitol, or condensation products derived from partial esters of fatty acids and hexitol anhydrides (for example, polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include but are not limited to lecithin and gum arabic. Known preservatives include but are not limited to methyl, ethyl or n-propyl-p-hydroxybenzoate, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickeners for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

[0304] The liquid solution of active ingredient in aqueous or oily solvent can be prepared in a substantially identical manner to liquid suspension, with the major difference being that the active ingredient is dissolved rather than suspended in a solvent. As used herein, "oily" liquid is a liquid comprising carbonaceous liquid molecules and showing polarity characteristics less than water. The liquid solution of the pharmaceutical composition of the present invention can comprise each component described about the liquid suspension, and it should be understood that suspending agents will not necessarily help the active ingredient to be dissolved in a solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin.

[0305] Powder and granular formulations of the pharmaceutical preparations of the present invention can be prepared using known methods. Such preparations can be directly administered to a subject, for example, to form tablets, fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Each of these preparations may also include one or more of a dispersant or wetting agent, a suspending agent, and a preservative. These preparations may also include additional excipients, such as fillers and sweeteners, flavorings, or coloring agents.

[0306] Pharmaceutical composition of the present invention can also be prepared, packaged or sold in the form of oil-in-water emulsion or water-in-oil emulsion. The oil phase can be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination of these oils. Such compositions can also include one or more emulsifying agents, such as naturally occurring gums such as gum arabic or tragacanth, naturally occurring phosphatides such as soybean or lecithin phospholipids, esters or partial esters such as sorbitan monooleate obtained by the combination of fatty acid and hexitol anhydride, and condensation products of this type of partial ester and ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions can also contain other ingredients, including for example sweeteners or flavorings.

[0307] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of depositing or binding the chemical composition onto a surface, methods of incorporating the chemical composition into the structure of the material during its synthesis (i.e., as with the use of physiologically degradable materials), and methods of absorbing aqueous or oily solutions or suspensions into an absorbent material, with or without subsequent drying.

[0308] The administration regimen may affect what constitutes an effective amount. The therapeutic formulation may be administered to a subject before or after diagnosis of the disease. In addition, several separate doses and staggered doses may be administered daily or sequentially, or the dose may be infused continuously, or may be a bolus injection. In addition, the dose of the therapeutic formulation may be increased or decreased in proportion to the urgency of the treatment or prevention situation.

[0309] Administration of the compositions of the present invention to subjects (including mammals, such as humans) can be carried out using known procedures, at doses and time periods that are effective for preventing or treating diseases. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary according to factors such as: the activity of the specific compound used; the time of administration; the excretion rate of the compound; the duration of treatment; other drugs, compounds or substances used in combination with the compound; the state of the disease or condition of the subject being treated, age, sex, weight, condition, general health and previous medical history, and similar factors well known in the medical field. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several separate doses may be administered daily or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. A non-limiting example of an effective dosage range for the therapeutic compound of the present invention is about 1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art will be able to study the relevant factors and make decisions about the effective amount of the therapeutic compound without excessive experimentation.

[0310] The compound may be administered to the subject several times a day, 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 few months or even once a year or less frequently. It should be understood that the amount of the compound administered daily may be administered every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days in a non-limiting example. For example, in the case of administration every other day, a 5 mg daily dose may be administered starting on Monday, a subsequent 5 mg daily dose may be administered for the first time on Wednesday, a subsequent 5 mg daily dose may be administered for the second time on Friday, and the like. The frequency of the dose will be apparent to those skilled in the art, and will depend on many factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like.

[0311] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention may be varied so as to obtain an amount of the active ingredient which achieves the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0312] A physician, such as an internist or veterinarian, having ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the compound of the invention used in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0313] In specific embodiments, it is particularly advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit dosages for subjects to be treated; each unit contains a predetermined quantity of the therapeutic compound calculated to produce the desired therapeutic effect and the required pharmaceutical vehicle. The dosage unit form of the present invention is determined by and directly dependent on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such therapeutic compounds for treating diseases in subjects.

[0314] In one embodiment, the composition of the present invention is applied to a subject in a dosage range of one to five or more times per day. In another embodiment, the composition of the present invention is applied to a subject in a dosage range including but not limited to once a day, once every two days, once every three days to once a week and once every two weeks. It will be readily understood by those skilled in the art that the frequency of applying the various combination compositions of the present invention will vary from subject to subject depending on a variety of factors, including but not limited to age, disease to be treated or illness, sex, general health and other factors. Therefore, the present invention should not be construed as being limited to any specific dosage regimen and the precise dosage and composition applied to any subject will be determined by the attending physician taking into account all other factors about the subject.

[0315] The compounds of the present invention for administration may range 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 therebetween.

[0316] In some embodiments, the dosage of the compounds of the invention is from about 1 mg to about 2,500 mg. In some embodiments, the dosage of the compounds of the invention used in the 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, the dose of the second compound as described herein (i.e., a drug used to treat the same disease as or another disease treated by the composition of the invention) 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.

[0317] In one embodiment, the invention relates 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 agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0318] The term "container" includes any receptacle for holding a pharmaceutical composition. For example, in one embodiment, the container is a package containing a pharmaceutical composition. In other embodiments, the container is not a package for holding a pharmaceutical composition, that is, the container is a receptacle for holding a packaged pharmaceutical composition or an unpackaged pharmaceutical composition and instructions for using the pharmaceutical composition, such as a box or a vial. In addition, packaging technology is well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be included in the package containing the pharmaceutical composition, so the functional relationship between the instructions and the packaged product increases. However, it should be understood that the instructions may contain information related to the ability of the compound to perform its intended function (e.g., to treat or prevent a subject's disease, or to deliver an imaging agent or diagnostic agent to a subject).

[0319] Routes of administration for any composition of the invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, and nasal), intravesical, intraduodenal, intragastric, rectal, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, or administration.

[0320] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pills, sera, lozenges, creams, pastes, ointments, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0321] Methods and treatment methods for regulating RNA cleavage and / or polyadenylation

[0322] In one aspect, the invention provides a method for regulating 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 complementary to a target RNA sequence in an RNA transcript, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in an RNA transcript.

[0323] In one embodiment, the subject is a cell. In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0324] In one embodiment, cleavage, polyadenylation, or both of an RNA transcript is modulated in vitro. In one embodiment, cleavage, polyadenylation, or both of an RNA transcript is modulated in vivo.

[0325] 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 complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence that is 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; and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0326] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence that is 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 that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0327] In one aspect, the invention provides a method for treating a disease or condition associated with abnormal cleavage and / or polyadenylation. 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, a cleavage and / or polyadenylation protein, and (2) a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0328] 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, a cleavage and / or polyadenylation protein, and (2) a guide nucleic acid molecule comprising a crRNA targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0329] In one embodiment, the gene is associated with abnormal 3'UTR extension, 3'UTR shortening, polyadenylation signal alteration or other abnormal polyadenylation causing a disease or condition.Exemplary diseases or conditions and corresponding targets include, but are not limited to, those listed in Table 1.

[0330] For example, in one embodiment, Alzheimer's disease is treated, wherein the method comprises administering to the subject (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 complementary 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.

[0331] Table 1. Diseases or disorders and target genes associated with aberrant cleavage and / or polyadenylation.

[0332]

[0333]

[0334]

[0335]

[0336] RNA reduction methods and treatments

[0337] In one aspect, the present invention provides a method for reducing the number of nuclear RNA in a subject. In one embodiment, the nuclear RNA is an abnormal nuclear RNA. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and an NLS or a fusion protein of the present 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 complementary to a target RNA sequence in the nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the nuclear RNA.

[0338] 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 SEQ ID NO:702; and a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence that is 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:731; and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0339] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence that is 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 NO:702; and a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0340] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal or fungal 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.

[0341] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0342] In one embodiment, the amount of nuclear RNA is reduced in vitro. In one embodiment, the amount of nuclear RNA is reduced in vivo.

[0343] In one embodiment, the nuclear RNA is a nuclear RNA focus. In one embodiment, the guide nucleic acid comprises a sequence complementary to the CTG repeat expansion in the 3'UTR of the human dystrophin myotonia-protein kinase (DMPK) gene. In one embodiment, the guide nucleic acid comprises the sequence of one of SEQ ID NOs: 762-764.

[0344] In one aspect, the present invention provides a method for treating a subject suffering from a disease or condition associated with abnormal nuclear RNA. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and an NLS or a fusion protein of the present 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 complementary to a target RNA sequence in nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA.

[0345] In one embodiment, the disease or disorder associated with abnormal nuclear RNA is selected from the group consisting of myotonic dystrophy type 2 (DM2), amyotrophic lateral sclerosis (ALS), Huntington's disease-like 2 (HDL2), spinocerebellar ataxia 8, 31 and 10 (SCA8, -31, -10), and fragile X-associated tremor ataxia syndrome (FXTAS).

[0346] In one embodiment, the abnormal nuclear RNA is a toxic nuclear RNA focus. In one embodiment, the disease or condition associated with the toxic nuclear RNA focus is myotonic dystrophy type 1. In one embodiment, the targeting nucleotide sequence comprises a sequence complementary to the CTG repeat expansion in the 3'UTR of the human dystrophic myotonia-protein kinase (DMPK) gene. In one embodiment, the targeting nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO: 762-764.

[0347] In one aspect, the invention provides a method for cleaving nuclear RNA 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 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 complementary to a target RNA sequence in nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA.

[0348] In one aspect, the present invention provides a method for treating a disease or condition associated with increased gene expression. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and an NLS, or a fusion protein of the present 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 complementary to a target RNA sequence in an RNA transcript of the gene, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in an RNA transcript of the gene. In one embodiment, the Cas protein cleaves the RNA transcript, thereby preventing translation and protein expression.

[0349] In one aspect, the present invention provides methods for treating diseases or conditions associated with RNA. For example, in one embodiment, the present invention provides a method for treating RNA viral infection. In one embodiment, the present invention provides a method for treating DNA viral infection. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and an NLS, or a fusion protein of the present 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 complementary to a target RNA sequence in a viral RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in a viral RNA. In one embodiment, the Cas protein cleaves RNA transcripts, thereby preventing the translation and expression of viral proteins.

[0350] Methods for visualizing RNA

[0351] In one aspect, the present invention provides a method for visualizing nuclear RNA in a subject. In one embodiment, the method includes (A) administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and a fluorescent protein or a fusion protein of the present invention comprising a Cas protein and a fluorescent protein, and (2) encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA; and (B) visualizing the nuclear RNA. In one embodiment, the RNA visualization occurs by imaging. In one embodiment, the Cas protein binds to a guide nucleic acid, which binds to a target RNA sequence and detects a fluorescent protein.

[0352] 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: 699-701; and a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence that is 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; and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0353] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence that is 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 that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0354] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal or fungal 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.

[0355] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0356] In one embodiment, nuclear RNA is visualized in vitro. In one embodiment, nuclear RNA is visualized in vivo.

[0357] In one embodiment, the nuclear RNA is a nuclear RNA focus. In one embodiment, the crRNA comprises a sequence complementary to the CTG repeat expansion in the 3'UTR of the human dystrophic myotonia-protein kinase (DMPK) gene. In one embodiment, the crNA comprises a sequence of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

[0358] In one embodiment, the present invention provides a method for diagnosing a disease or condition associated with abnormal nuclear RNA. In one embodiment, the method comprises administering (A) administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and a fluorescent protein or a fusion protein of the present 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 complementary to a target RNA sequence in nuclear RNA or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in abnormal nuclear RNA; (B) visualizing the abnormal nuclear RNA; and (C) diagnosing the disease or condition when the genetic nuclear RNA is present.

[0359] Treatment and use

[0360] The present invention provides methods for treating a disease or condition in a subject, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition. For example, in one embodiment, the methods of the present invention treat a disease or condition in a mammal, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a plant, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a yeast organism, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition.

[0361] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal or fungal 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.

[0362] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0363] In one embodiment, the disease or condition is caused by one or more mutations in a genomic locus. Thus, in one embodiment, the disease or condition may be treated, alleviated, or risk reduced by an element that prevents or reduces mRNA transcripts or prevents or reduces translation of proteins. Thus, in one embodiment, the method comprises manipulation of RNA transcripts.

[0364] In one embodiment, the disease or condition is caused by abnormal RNA. Thus, in one embodiment, the disease or condition can be treated, alleviated, or risk reduced by preventing or reducing elements of RNA transcripts. Thus, in one embodiment, the method comprises manipulation of RNA transcripts.

[0365] 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, a cleavage and / or polyadenylation protein, and (2) a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the cleavage and / or polyadenylation protein regulates cleavage and / or polyadenylation of the RNA transcript.

[0366] 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 complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence that is 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; and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0367] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence that is 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 that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0368] In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present invention comprising a Cas protein and an NLS, or a fusion protein of the present 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 complementary to a target RNA sequence in nuclear RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA.

[0369] 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 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 complementary to a target RNA sequence in nuclear RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in nuclear RNA. In one embodiment, the Cas protein cleaves the RNA transcript.

[0370] 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 SEQ ID NO:702; and a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in a gene, wherein the gene encodes an RNA transcript. In one embodiment, the method comprises administering to the subject a nucleic acid molecule comprising a nucleic acid sequence that is 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:731; and a nucleic acid sequence encoding a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0371] In one embodiment, the method comprises administering to the subject a fusion protein comprising an amino acid sequence that is 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 NO:702; and a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a target region in a gene, wherein the gene encodes an RNA transcript.

[0372] In one embodiment, the disease or condition is associated with RNA. For example, in one embodiment, the disease or condition 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 strand RNA virus.

[0373] 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 picornavirus, navirus), Colorado tick fever virus, Changuinola virus, Oshima virus, Lebombo virus, Orungo virus, Rotavirus A, Rotavirus B, Rotavirus C, Banna virus, Borna disease virus, Lake Victoria Marburg virus, Reston Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, 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, Piri virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis Indiana virus, Vesicular stomatitis New Jersey virus,Australian bat lyssa virus, Duvenhage virus, European bat lyssa virus 1, European bat lyssa virus 2, Mokola virus, rabies virus, Guanarito virus, Junin virus, Lassa virus, lymphocytic choriomeningitis virus, Machupo virus, Pichinde virus, Sabia virus, Whitewater Arroyo virus, Bunyoro virus, Bowamba virus, California encephalitis virus, Caraparu virus, Catu virus, Guama virus, Guaru virus, Kaili virus, Marituba virus, Oriboca virus, Oropouche virus, Shuni virus, Takaema virus, Wyomea virus, Andes virus , Naganuma virus, Kurokogou virus, Dobrava-Belgrade virus, Hanta virus, Laguna Negra virus, New York virus, Puumala virus, Seoul virus, Sin Nombre virus, Crimean-Congo hemorrhagic fever virus, Dube virus, Candiru virus, Ponta Toru virus, Rift Valley fever virus, Naples sandfly fever virus, influenza A virus, influenza B virus, influenza C virus, Tori virus, Togotu virus, hepatitis D 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, Theiler virus, equine rhinitis A virus, foot-and-mouth disease virus, hepatitis A virus, human parechovirus, Yongan River 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, Aluo virus, Banzi virus, dengue virus, Ilheus virus, Japanese encephalitis virus, Kokobella virus, Kosanur Forest disease virus, jumping disease virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, Powassan virus, Gerberwein virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Usutu virus, Wessels Brown virus, West Nile virus, Yellow fever virus, Zika virus, Hepatitis C virus, Hepatitis E virus, Bama Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Evogre virus, Geta virus, Mayaro virus, Mukumbo virus, Anionyan virus, Napishuna virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Huataroua virus, Rubella virus.

[0374] In one embodiment, the DNA virus is a single-stranded DNA virus or a double-stranded DNA virus. Exemplary DNA viruses include, but are not limited to, AAV9, AAV12, AAV18, AAV31, HBV, EBV, KSHV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV45, Merkel cell polyomavirus, and MCV.

[0375] In one embodiment, the disease or condition is associated with aberrant polyadenylation or aberrant RNA. In one embodiment, the method treats a disease or condition associated with aberrant polyadenylation or aberrant RNA, wherein the aberrant polyadenylation or aberrant RNA is targeted.

[0376] In one embodiment, the disease or condition is an endocrine disease. For example, in one embodiment, endocrine diseases include but are not limited to β-thalassemia, neonatal diabetes,

[0377] IPEX syndrome, Mayer–Rokitanski–Küster–H auser syndrome, hypothalamic-pituitary-adrenal axis disorder, adrenal dysfunction, gonadal dysfunction, ectopic Cushing's syndrome, preeclampsia, diabetic nephropathy, type 1 diabetes mellitus, type 2 diabetes mellitus, and IGF-1 deficiency.

[0378] In one embodiment, the disease or condition is a neoplastic disease. For example, in one embodiment, neoplastic diseases include, but are not limited to, mantle cell lymphoma, hereditary and sporadic parathyroid tumors, medullary thyroid carcinoma, proliferative disorders, colorectal cancer, glioblastoma, chronic lymphocytic leukemia, and breast cancer.

[0379] In one embodiment, the disease or disorder is a nervous system disease or disorder. For example, in one embodiment, nervous system diseases include, but are not limited to, Parkinson's disease, oculopharyngeal muscular dystrophy, Huntington's disease, Fabry disease, fragile X syndrome, spinal muscular atrophy, amyotrophic lateral sclerosis, 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.

[0380] 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 beta-thalassemia and alpha-thalassemia.

[0381] In one embodiment, the disease or condition is an infection or immune disease or condition. For example, in one embodiment, an infection or immune disease includes but is not limited to B cell differentiation, T cell activation, systemic lupus erythematosus, Wiskott-Aldrich syndrome, osteoarthritis, scleroderma, and IPEX syndrome.

[0382] In one embodiment, the disease or disorder is a musculoskeletal disease or disorder. For example, in one embodiment, the infectious or immune disease includes myotonic dystrophy type 1, spinal and bulbar muscular atrophy, and dentatorubral pallidum Lewy body atrophy.

[0383] 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), which are incorporated herein by reference in their entirety.

[0384] Table 2. Diseases or conditions and target genes

[0385]

[0386]

[0387]

[0388]

[0389] Amino acid and nucleic acid sequences

[0390] Table 3 provides references to the sequences.

[0391] Table 3

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414] Experimental Examples

[0415] The present invention will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Therefore, the present invention should not be interpreted in any way as being limited to the following examples, but should be interpreted as covering any and all variations that become apparent due to the teachings provided herein.

[0416] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the present invention and practice the claimed methods. Therefore, the following working examples particularly point out certain embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0417] Example 1: Targeted cleavage and polyadenylation of RNA by CRISPR-Cas13

[0418] The data presented here demonstrate that fusion of a catalytically dead Cas13 to a single mammalian polyadenylation factor, Nudix hydrolase 21 (NUDT21), allows site-specific CRISPR-Cas13-guided RNA cleavage and polyadenylation in mammalian cells. This approach is termed Postscriptr and can be used for non-genomic manipulation of gene expression and may have potential future therapeutic applications for treating human RNA processing diseases.

[0419] The 3' site of RNA cleavage and the addition of the poly (A) tail are precisely determined by the intrinsic polyadenylation signal (PAS) sequence, which is usually composed of the canonical hexameric motif AAUAAA and upstream and downstream sequence elements (USE and DSE, respectively) (Tian and Graber, Wiley Interdiscip Rev RNA, 2012, 3: 385-396). The AAUAAA PAS motif is usually located about 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 protein 33 containing WD repeat sequences (WDR33) (Chan et al., Genes Dev, 2014, 28: 2370-2380). The components of the cleavage factor Im (CFIm) complex directly bind to the USE motif UGUA, which is located about 50 nucleotides upstream of the RNA cleavage site (Ellkon et al., Nat Rev Genet, 2013, 14: 496-506). In transcripts lacking the canonical AAUAAA motif, the CFIm complex serves as the main determinant of poly (A) signal recognition (Venkataraman et al., Genes Dev, 2005, 19: 1315-1327). Nudix hydrolase 21 (NUDT21 / CPSF5) (RNA binding component of the CFIm complex) serves as an activator of 3' end processing and a regulator of selective polyadenylation site selection (Zhu et al., Mol Cell, 2018, 69: 62-74). The binding of the CFIm complex is the first step in 3' end processing and is used to recruit additional cofactors to the 3' end processing machinery. Direct interaction between components of the polyadenylation supercomplex and the C-terminal domain of RNA polymerase II triggers disassembly of the elongation complex and coordinates 3'-end processing with transcription termination (Zhao et al., Microbiol Mol Biol Rev, 1999, 63:405-45).

[0420] Cas13 is a bacterial-derived crRNA-guided endonuclease with specific affinity for RNA (Abudayyeh et al., Science, 2016, 353: aaf5573). Similar to Cas9, residue mutations within the nuclease domain of Cas13 produce catalytic dead enzymes, but retain RNA binding affinity (dCas13). Recently, fusion of dCas13 with 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 use dCas13 to direct polyadenylation complex formation, three fusion proteins were designed that combined catalytically dead PspCas13b (dPspCas13b) with RNA-binding components of the human 3′-end processing machinery, including CPSF30, WDR33, and NUDT21 ( Figure 1 A). 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)] was included between dPspCas13b and the polyadenylation component to reduce the possibility of steric hindrance.

[0421] Unlike RNA modification applications using dCas13 that occur in the cytoplasm, post-transcriptional cleavage and polyadenylation of RNA occur in the eukaryotic nucleus and therefore require efficient nuclear localization of dCas13 fusion proteins. The large size of Cas13 and its lack of an intrinsic nuclear localization signal (NLS) prevent efficient nuclear localization in mammalian cells. Consistent with this, when expressed in mammalian COS7 cells, dPspCas13b fusion proteins lacking mammalian NLS were retained in the cytoplasm, as detected by immunocytochemistry using an anti-FLAG antibody ( Figure 5 Surprisingly, addition of either the canonical SV40 NLS or a bipartite NLS from nucleoplasmin (NPM) was not sufficient to promote nuclear localization of dPspCas13b fusion proteins ( Figure 5 ). However, addition of a single copy of a non-canonical bipartite NLS derived from the yeast Ty1 retrotransposon resulted in robust nuclear localization of all three dPspCas13b fusion proteins ( Figure 1 B), which utilizes the normal cellular import machinery but contains a linker sequence that is almost three times longer than a typical bipartite NLS (McLane et al., NAR, 2008, 36:4317-26; Kenna et al., Mol Cell Biol, 1998, 18:1115-24).

[0422] Next, we designed a reporter gene to detect alternative cleavage and polyadenylation in mammalian cells, which can switch between fluorescent and non-fluorescent open reading frames of superfolded GFP (sfGFP) ( Figure 2 sfGFP forms a β barrel consisting of 11 antiparallel β strands, which can tolerate sequence insertion between the 10th and 11th β strands, but loses fluorescence if the 11th β strand is removed (Kamiyama et al., Nat Commun, 2016, 7: 11046; Feng et al., Nat Commun, 2017, 8: 370). The sfGFP reporter gene construct was generated with the coding sequence of the 11th β strand embedded in a prototype mammalian intron (the second intron of the rabbit β globin gene) (sfGFPapa) ( Figure 2 A). The coding sequence of the 11th β-chain was designed in frame with the upstream sfGFP coding sequence, so that translation of the proximal open reading frame would encode the complete sfGFP sequence, despite the presence of a 14-amino acid linker sequence between the 10th and 11th β-chains resulting from translation of the intervening intron sequence [sfGFP(1-10)-L-11]. Expression of the sfGFPapa reporter gene in mammalian COS7 cells resulted in almost no detectable fluorescence, indicating efficient removal of the intron containing the 11th β-chain and translation of the open reading frame encoding the non-fluorescent sfGFP(1-10) protein ( Figure 2 B to Figure 2 D). However, cells expressing the sfGFPapa reporter gene treated with the splicing inhibitor isoginkgetin (O'Brien et al., J Biol Chem, 2008, 283:33147-54) produced detectable green fluorescence after 24 hours, demonstrating that the modified sfGFP open reading frame containing a linker between the 10th and 11th β-strands is functional ( Figure 6 A and Figure 6 B). Insertion of the SV40 polyadenylation cassette downstream of the coding sequence for the 11th β-strand of sfGFP (sfGFPapa-pA) resulted in robust green fluorescence, demonstrating that intron cleavage and polyadenylation of the sfGFPapa reporter gene promotes utilization of a functional sfGFP(1-10)-L-11 open reading frame ( Figure 2 E to 2H). Taken together, these data demonstrate that the sfGFPapa reporter gene encodes functional and non-functional open reading frames that can be switched in response to changes in splicing or intron cleavage and polyadenylation.

[0423] To determine whether the dPspCas13b fusion protein could promote CRISPR-Cas13-mediated cleavage and polyadenylation of the sfGFPapa reporter gene mRNA, a crRNA targeting the intronic sequence downstream of the coding sequence of the 11th β-strand of sfGFP in the sfGFPapa reporter gene was designed ( Figure 3 A). In live cell assays, no fluorescence signal was detected in cells expressing any dPspCas13b fusion protein using non-targeting crRNA relative to the sfGFPapa reporter alone ( Figure 3 B and Figure 7 However, expression of intron-targeted crRNA with dPspCas13b-NUDT21 fusion protein, but not CPSF30 or WDR33 fusion proteins, resulted in detectable green fluorescent cells after 24 h ( Figure 3 B and Figure 7 ).

[0424] 3' Rapid Amplification of cDNA Ends (3' RACE) was performed to determine whether the fluorescent 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 poly(T) oligonucleotides 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 3 C). Interestingly, in cells targeted with intronic crRNA, another broad band of smaller RACE products was detected, suggesting that cleavage and polyadenylation may have occurred near the targeting crRNA sequence ( Figure 3 C and Figure 3 D). To determine the sequence of the 3' RACE product, a region of 200-500 base pairs was gel purified and subcloned using TOPO TA cloning. Eleven unique clones were isolated and sequenced, which revealed that sfGFPapa mRNA was cleaved and polyadenylated at sites ranging from -7 to +110 nucleotides on the 3' side relative to the crRNA target sequence ( Figure 3 E). In contrast to mammalian cleavage, which occurs primarily after CA dinucleotides, PostscriptR-induced cleavage most often occurs at C or T nucleotides ( Figure 3F). Interestingly, 4 of the 11 clones showed 3' addition of non-templated nucleotides before poly(A) tail extension, which is considered rare in mammalian species but ubiquitous in plant mRNAs (Jin and Bian, RNA, 2004, 10: 1695-97). These data demonstrate that the dPspCas13b-NUDT21 fusion protein is sufficient to induce cleavage and polyadenylation of the reporter mRNA at the intron sequence targeted by the crRNA.

[0425] To determine whether Postscriptr could promote the selective cleavage and polyadenylation of endogenously expressed human mRNAs, transcripts encoding human sterol regulatory element binding protein 1 (SREBP1) were targeted. SREBP1 is a ubiquitously expressed transcription factor that transactivates genes containing sterol regulatory elements (SREs) and encoding proteins that control lipid synthesis and uptake, such as the low-density lipoprotein receptor (LDLR) ( Figure 4 )(Horton et al., J Clin Invest, 2002, 109: 1125-31). SREBP1 is first synthesized as a transcriptionally inactive precursor protein anchored in the membrane of the endoplasmic reticulum (ER) (Brown and Goldstein, Cell, 1997, 89: 331-40) ( Figure 4 A). In sterol-depleted cells, proteolytic cleavage of SREBP1 by S2P releases the N-terminal DNA-binding fragment, which allows SREBP1 to enter the nucleus and activate gene transcription (Brown et al., Cell, 2000, 100:391-98) ( Figure 4 A). SREBP1 transcripts in the liver utilize an endogenous intronic PAS and produce protein fragments [SREBP1aΔ, AB373958, SREBP1cΔ, AB373959] that terminate at a site adjacent to the transcriptionally active S2P cleavage product ( Figure 4 B and C). Next, we determined whether Postscriptr could promote the utilization of the SREBP1 intronic PAS and upregulate SREBP1 target gene expression in non-liver cell lines. The dPspCas13b-NUDT21 fusion protein was co-expressed with SREBP1-targeting or non-targeting crRNA in human HEK293T cells ( Figure 4 D). When 3'RACE was performed, a strong band was detected corresponding to the approximate size of the predicted 3'RACE product in cells transfected with SREBP1 targeting crRNA, and no band was detected in cells transfected with control crRNA ( Figure 8 Direct sequencing of this 3' RACE product revealed that the SREBP1 transcript is cleaved and polyadenylated 71 nucleotides downstream of the crRNA target sequence, and that splicing of the terminal exon is inhibited ( Figure 4 D and E). Cleavage occurs at the CA dinucleotide 36 nucleotides downstream of the reported cleavage site in humans and generates an open reading frame that terminates at a stop codon within the retained intronic sequence, thereby adding an additional 16 amino acids to the C-terminus of SREBP1 ( Figure 4 E and F). Gene expression analysis in mixed populations of SREBP1-targeted and non-targeted HEK293T cells showed no change in overall SREBP1 transcript levels when using qRT-PCR and PCR primers upstream of the crRNA target sequence ( Figure 4 G). However, primers spanning the targeted exon showed that SREBP1 transcript levels were significantly reduced in targeted cells compared with non-targeted cells, indicating that transcription termination can be coupled to Postscriptr-mediated cleavage and polyadenylation ( Figure 4 H). Interestingly, in SREBP1-targeted cells, a significant increase in the transcription level of the well-characterized SREBP1-responsive gene LDLR was further detected ( Figure 4 I).

[0426] In summary, the date provided herein demonstrates the development of a unique CRISPR-Cas13 RNA modification technology to induce sequence-specific cleavage and polyadenylation of RNA in mammalian cells. This approach utilizes a dPspCas13b-NUDT21 fusion protein, which, as shown herein, requires a non-classical bipartite NLS for nuclear localization in mammalian cells. The data provided herein demonstrate that the dPspCas13b-NUDT21 fusion protein is sufficient to promote cleavage and polyadenylation at intronic sequences within the novel gain-of-function fluorescent reporter gene sfGFPapa. Given that the second intron of the rabbit β-globin gene does not contain any known endogenous PAS sequences, the ability of Postscriptr to promote cleavage and polyadenylation of the sfGFPapa reporter gene is striking. These findings demonstrate that the dPspCas13b-NUDT21 fusion protein can independently promote the recruitment of a functional 3' end processing complex at the crRNA targeting sequence. These findings support a role for NUDT21 as an emerging master regulator of cleavage and alternative polyadenylation site selection in mammals (Zhu et al., Mol Cell, 2018, 69:62-74; Brumbaugh et al., Cell, 2018, 172:106-120; Sartini et al., Biol Reprod, 2008, 89:472-82).

[0427] It is not clear why the fusion of dPspCas13b with CPSF30 or WDR33 is not sufficient to promote the cleavage and polyadenylation of the sfGFPapa reporter gene in these assays. Since both CPSF30 and WDR33 have been shown to be in direct contact with the AAUAAA PAS motif, one possibility may be that PAS RNA is required to promote ternary complex formation. Alternatively, the tethering of CPSF30 and WDR33 may not be sufficient to induce 3' end processing, a possibility consistent with data showing that the insertion of a minimal synthetic PAS (including AAUAAA motifs and downstream sequence elements) in the second intron of the rabbit β-globin gene reporter gene is not sufficient to promote intron cleavage and polyadenylation (Levitt et al., Genes Dev, 1989 3: 1019-25).

[0428] This article also demonstrates that Postscriptr can direct cleavage and polyadenylation at the endogenous intronic PAS of the human SREBP1 gene. It is not clear why a single primary cleavage site is induced when targeting the endogenous SREBP1 intronic PAS, but a series of cleavage products spanning approximately 100 nucleotides are induced when targeting the sfGFPapa reporter gene. Although not wishing to be bound by any particular theory, it is possible that the lack of an intrinsic PAS sequence within the second intron of the rabbit β-globin gene in the sfGFPapa reporter gene may be required to help guide cleavage site specificity. Future studies are necessary to describe minimal upstream and downstream sequence elements that can enhance Postscriptr targeting at ectopic target sites.

[0429] Although the endonuclease activity of Cas13 is very effective for gene knockdown in mammalian cells (Abudayyeh et al., Nature, 2017, 550: 280-84), Postscriptr technology allows a unique method to manipulate RNA in cells, which may help study the manipulation of 3' end RNA processing and endogenous gene expression. An interesting example will be the inquiry into lncRNA function, many of which control gene expression by recruiting chromatin modification complexes or by establishing a permissive local chromatin environment (Anderson et al., Nature, 2016, 539: 433-36; Joung et al., Nature, 2017, 5448: 343-46; Engreitz et al., Nautre, 2016, 539: 452-55; Rinn and Chang, Annu Rev Biochem, 2012, 81: 145-66). Postscriptr-guided cleavage, polyadenylation, and transcription termination of lncRNAs can allow identification of functional lncRNA domains or functional genomic regions. In addition, many human diseases are the direct result of loss-of-function mutations within the poly(A) signal sequence, which can lead to transcript elongation as well as mRNA stability and translation (Higgs et al., Nature, 1983, 306:398-400; Orkin et al., EMBO J, 1985, 4:453-56; Rund et al., PNAS, 1992, 89:4324-28). Here, the unique therapeutic application of Postscriptr technology may help correct normal RNA cleavage and polyadenylation site selection in diseased tissues.

[0430] Synthetic DNA and cloning

[0431] The coding sequences of human CPSF30, WDR33 and NUDT21 were designed and synthesized for assembly into gBlocks (IDT, Integrated DNA Technologies) and cloned into a modified version of the CS2 mammalian expression vector containing a 5'T7 promoter as a C-terminal fusion with dPspCas13b (CSX). The sfGFPapa reporter gene was designed and synthesized as two separate gBlock gene fragments and subcloned into the CS2 mammalian expression vector. All plasmid coding sequences were fully verified by Sanger sequencing. For a list of coding sequences and reporter gene sequences, see Example 3.

[0432] Cell lines, transient transfection, and drug treatment

[0433] COS7 cell line was maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin at 37°C in an atmosphere of 5% CO2. Cells were inoculated and transiently transfected using Fugene6 (Promega) according to the manufacturer's protocol. 20 hours after transfection, cells were imaged, or treated with DMSO or isoginkgetin and cultured for another 24 hours before imaging. For live cell imaging, cells were washed twice with DPBS, counterstained with 10 μg / ml Hoechst 33342 (Invitrogen) in DPBS for 10 minutes at room temperature, and imaged on a ZOE fluorescent cell imager (Bio-Rad).

[0434] Antibodies and immunohistochemistry

[0435] Transiently transfected COS7 cells were fixed in 4% formaldehyde in PBS for 20 minutes, blocked in 3% bovine serum albumin (BSA), and incubated with the primary anti-FLAG (Sigma, F1864) at 1:1000 in 1% BSA for 1 hour at room temperature. The cells were then incubated with Alexa488-conjugated secondary antibodies (Thermofisher) in 1% BSA for 30 minutes at room temperature. Coverslips were mounted with anti-fading fluorescent mounting medium containing DAPI (Vector Biolabs, H-1200) and imaged with a confocal microscope.

[0436] 3' Rapid Amplification of cDNA Ends (3'RACE)

[0437] The transfected cells were washed twice with DPBS and harvested in 1 ml of Trizol to isolate total RNA. RACE-compatible cDNA was generated using SuperScript III (Invitrogen) according to the manufacturer's protocol, except for a custom oligo d(T) oligonucleotide containing two 5' nested primer sequences. RACE PCR was performed using two nested target gene-specific primers and a touchdown PCR protocol as follows: 94°C for 1 minute (1 cycle); 94°C for 30 seconds, 72°C for 1 minute (5 cycles); 94°C for 30 seconds, 70°C for 4 minutes (5 cycles); 94°C for 20 seconds, 68°C for 1 minute (25 cycles); 72°C for 5 minutes. See Example 3 for a list of PCR primers and 3' RACE-specific primers.

[0438] Example 2: Enhanced destruction of poly(T) sequences in the direct repeat sequence of PspCas13b crRNA Postscript activity

[0439] The CRISPR-Cas13 system is naturally guided by small CRISPR RNAs (crRNAs), which are typically expressed in mammalian cells from polymerase III (Pol III) promoters such as U6, 7SK, or H1. Unlike Pol II-dependent polymerases, Pol III promoters are terminated by short poly(T) sequences, typically consisting of only 6 or more consecutive Ts. Detailed analysis of the termination specificity of Pol III promoters in vitro and in mammalian cells revealed that even a short stretch of 4 Ts resulted in a 75% reduction in full-length expression of the small RNA ( Fig. 9 A) (Gao et al., Mol Ther Nucleic Acids, 2018, 10: 36-44). Many Cas13b family members contain 4-5 internal Ts in their direct repeat (DR) sequences, which can significantly prevent their efficient expression from Pol III-driven promoters and inhibit Cas13b targeting ( Fig. 9 B) It is not clear whether these sequences are located in the DR loop region ( Fig. 9 C) is essential for Cas13b targeting. Recent high-resolution crystal structures of PbuCas13b and BzoCas13b show that at least one T encodes a U that is directed away from the Cas13b protein, suggesting that these nucleotides at these positions may not be required for specific interactions ( Fig. 9 D). To determine whether the poly(T) sequence in the PspCas13b DR could be disrupted to enhance crRNA expression, conservative (U to C, pyrimidine to pyrimidine) single mutations were generated to determine their effects on Postscriptr (dPspCas13b-NUDT21) targeted activation of the sfGFPapa fluorescent reporter gene ( Fig. 9 E). It is noteworthy that mutations of nucleotides at both sites, T17C or T18C, resulted in enhanced Postscriptr activity, while T19C mutations eliminated activity. These results suggest that enhanced activity may result from increased crRNA production, while mutations at some positions may inhibit normal Cas-crRNA binding.

[0440] Example 3: Regulation of Postscriptr fusion proteins and effects on targeted RNA cleavage and polyadenylation

[0441] The NUDT21 polyadenylation factor is highly conserved among distantly related species. Multiple protein sequence alignment of NUDT21 orthologs from humans, flies, and worms revealed strong sequence identity across species ( Fig.10 ).

[0442] Notably, NUDT21 orthologs from distantly related species showed similar activation of the sfGFPapa reporter gene in mammalian cells in the context of a Postscriptr (dPspCas13b-NUDT21) fusion protein ( Fig.11 ).

[0443] Although these orthologs have considerable sequence identity, they show little conservation within the first approximately 30 N-terminal amino acids ( Fig.10 ). This region can act as a negative regulatory region of human NUDT21, so when the residues in this region are acetylated, the interaction between poly (A) polymerase (PAP) and the CF1m complex is inhibited (Shimazu et al., J Biol Chem, 2007, 282: 4470-78). Consistent with this effect, in the context of the Postscriptr fusion protein, the deletion of the N-terminal 30 amino acids of human NUDT21 is not essential for activity, but leads to enhanced activation of the sfGFPapa reporter gene.

[0444] Although NUDT21 normally targets RNA polyadenylation sequences by direct RNA binding, mutations of residues that eliminate 99% of NUDT21 RNA binding (R63S or F103A) have little effect on Postscriptr activity in the context of Postscriptr fusion proteins targeted to RNA by dCas13 (Yang et al., PNAS, 2010, 107:10062-67; Yang et al. Structure, 2011, 19:368-77).

[0445] Since NUDT21 acts as an obligate dimer, a Postscriptr fusion protein encoding an obligatory tandem dimer of NUDT21 was generated, which strikingly resulted in a significantly enhanced activation of the sfGFPapa reporter gene.

[0446] Consistent with previous reports showing that significant truncation of the C-terminus of dPspCas13b had little effect on RNA targeting (Cox et al., Science, 2017, 358: 1019-27), similar deletions in the context of the Postscriptr fusion protein had little effect on Postscriptr activity but may be beneficial in reducing the overall size of the fusion protein for viral delivery via AAV.

[0447] Example 4: Postscriptr-Cas13-mediated prevention of myotonic dystrophy

[0448] Myotonic dystrophy (DM) is an autosomal dominant genetic disease characterized by progressive muscle weakness, muscle loss, myotonia, cardiac arrhythmias, and cognitive impairment. DM is the most common form of adult-onset muscular dystrophy and affects approximately 1 in 8,000 people. In some regions, DM may occur in 1 in 500 people.

[0449] There are two types of DM, DM1 and DM2, which are the result of nucleotide repeat expansions in the DMPK or CNBP genes, respectively. DM1 is more severe and more common and occurs due to trinucleotide (CUG) repeat expansions in the noncoding, 3' untranslated region (3'UTR) of the DMPK gene on human chromosome 19. DMPK RNA is transcribed in individuals with CUG repeat expansions (greater than about 50 copies) resulting in the formation of toxic nuclear RNA foci that bind and sequester RNA splicing factors of the myonecrotic nuclease-like (MBNL) family ( Fig.12 This results in loss of MBNL function and widespread splicing abnormalities. Although the coding sequence of DMPK is normal, nuclear RNA foci formed by abnormal DMPK transcripts also lead to reduced DMPK expression.

[0450] There is currently no cure for DM. Current treatments use antisense oligonucleotides specific for repeat expansions to prevent the binding and sequestration of myoblastoid protein. Alternatively, knockdown of mutant DMPK transcripts can prevent the accumulation of toxic repeat RNA, however, will result in loss of normal DMPK expression and function. Unfortunately, the delivery and administration of these oligonucleotide-based therapies remain major challenges for human patients.

[0451] As described in Example 1, Postscriptr allows targeted RNA cleavage and polyadenylation of RNA transcripts by CRISPR-Cas13. The Postscriptr method utilizes a fusion protein between the catalytically inactive Cas13 (dPspCas13b) and the mammalian polyadenylation factor NUDT21 ( Fig.13 A). Targeting the Postscriptr fusion protein using a guide RNA is sufficient to induce RNA cleavage and polyadenylation (Example 1). Therefore, Postscriptr-directed polyadenylation of the DMPK transcript will allow normal expression of the DMPK coding sequence and prevent transcription of downstream toxic RNA repeats ( Fig.13 B) Postscriptr technology can be delivered using AAV or other emerging viral delivery methods for long-term expression in somatic cells of DM1 individuals. Importantly, because Postscriptr technology targets only DMPK RNA, there is no potential for “off-target” DNA genome editing or potential for germline transmission.

[0452] The terminal exon of the human DMPK transcript is 923 nucleotides in length and contains the terminal coding sequence and the entire 3'UTR. Normal cleavage and polyadenylation occur downstream of the conserved consensus PAS (AAUAAA (SEQ ID NO: 789)) sequence. The CUG trinucleotide repeat expansion present in human DM1 patients occurs within the non-coding 3'UTR region of the terminal exon of the human DMPK gene, approximately 200 nucleotides downstream of the stop codon ( Fig.12 ).

[0453] Since DMPK transcripts are expressed in human HEK293T cells, it was determined whether Postscriptr was able to cleave and polyadenylate endogenous DMPK transcripts in this human cell line. The crRNA was designed to target the terminal exon of human DMPK, located upstream of the CUG repeat amplification site. Postscriptr fusion protein and crRNA were co-expressed into HEK293T cells for 24 hours, and total RNA was isolated for subsequent analysis by 3' rapid amplification of cDNA ends (RACE) using custom poly(T) oligonucleotides. PCR-based amplification using two nested DMPK-specific primers showed that one crRNA specifically induced the expression of a polyadenylated transcript that was shorter than the full-length 3'UTR (DMPK-PS) ( Fig.13 C). Sequencing of this band revealed that it was specific for the DMPK 3'UTR and was cleaved and polyadenylated 70 nucleotides downstream of the crRNA target sequence ( Fig.13 D). This corresponds to approximately 130 nucleotides upstream of the CUG repeat amplification site. Interestingly, the band corresponding to the full-length DMPK exon (DMPK-FL) was reduced in Postscriptr-modified cells ( Fig.13 D). Despite the presence of a conserved PAS sequence in this region, some human DMPK mRNAs have been reported to have similar proximal cleavage and polyadenylation sites. These data suggest that Postscriptr is sufficient to direct premature targeted cleavage and polyadenylation of human DMPK transcripts, which may be aided by endogenous cryptic upstream or downstream polyadenylation sequence elements. Since Postscriptr editing leads to premature termination of DMPK transcripts, this approach may be therapeutically useful for treating DM1.

[0454] Example 5: Targeted selective cleavage and polyadenylation of human DMPK transcripts

[0455] Myotonic dystrophy type 1 (DM1) is a monogenic autosomal dominant genetic disease characterized by progressive muscle atrophy, myotonia, arrhythmias and cognitive dysfunction. DM1 is the most common adult-onset muscular dystrophy and is caused by the amplification and expression of CUG trinucleotide repea...

Claims

1. A fusion protein comprising: a) CRISPR-associated (Cas) proteins, wherein: The Cas protein is dPspCas13b comprising a sequence selected from SEQ ID NOs: 47-48; b) a cleavage or polyadenylation protein, wherein the cleavage or polyadenylation protein is NUDT21; c) a nuclear localization signal (NLS) derived from the Ty1 retrotransposon; and d) joints; Among them, the fusion protein is composed of NLS, dPspCas13b, linker and NUDT21 from N-terminus to C-terminus.

2. The fusion protein according to claim 1, wherein The NUDT21 comprises a sequence selected from SEQ ID NOs: 51-58.

3. The fusion protein according to claim 1, wherein The NLS comprises a sequence selected from SEQ ID NOs: 83-695.

4. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises the sequence of SEQ ID NO:

698. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 4.

6. Use of the fusion protein of any one of claims 1 to 4 or the nucleic acid molecule of claim 5 and the guide nucleic acid in the preparation of a medicament for regulating cleavage and / or polyadenylation of RNA transcripts in a subject with myotonic dystrophy type 1, wherein The guide nucleic acid comprises a sequence that is complementary to a target RNA sequence in the RNA transcript.

7. The use according to claim 6, wherein The modulating cleavage and / or polyadenylation of an RNA transcript in a subject having myotonic dystrophy type 1 is performed in vitro or in vivo.

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