EP4665848A2 - Compositions and methods for treating motor neuron diseases - Google Patents

Compositions and methods for treating motor neuron diseases

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Publication number
EP4665848A2
EP4665848A2 EP24757590.5A EP24757590A EP4665848A2 EP 4665848 A2 EP4665848 A2 EP 4665848A2 EP 24757590 A EP24757590 A EP 24757590A EP 4665848 A2 EP4665848 A2 EP 4665848A2
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EP
European Patent Office
Prior art keywords
fold
cell
c9orf72
effector domain
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP24757590.5A
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German (de)
French (fr)
Inventor
X. Shawn Liu
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of EP4665848A2 publication Critical patent/EP4665848A2/en
Pending legal-status Critical Current

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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • ALS Amyotrophic lateral sclerosis
  • the GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) mutation in the noncoding region of the Chromosome 9 Open Reading Frame 72 (C9ORF72) gene is the most common genetic cause of ALS. In healthy individuals, the size of the hexanucleotide sequence is less than 24 repeats, but affected individuals have expansion mutations that can number in the thousands of repeats. Meijboom et al. CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Nat. Commun.13, 6286 (2022).
  • C9orf72 mutant carriers develop ALS and a significant portion of mutant carriers are asymptomatic, suggesting that other factors modify the disease onset such as DNA methylation.
  • hypermethylation of the G4C2 repeats and the C9orf72 promoter region were observed in C9orf72 mutant carriers.
  • the HRE is also the most common genetic cause of frontotemporal dementia (FTD). Both ALS and FTD are aggressive diseases with no treatments that significantly slow disease progression or extend life expectancy.
  • FTD frontotemporal dementia
  • Both ALS and FTD are aggressive diseases with no treatments that significantly slow disease progression or extend life expectancy.
  • C9ORF72 interacts with endosomes and is required for normal vesicle trafficking, autophagy induction and lysosomal biogenesis in diverse cell types, including motor neurons.
  • the HRE gives rise to three pathological hallmarks of C9ORF72 ALS: (1) It impairs expression, leading to C9ORF72 haploinsufficiency that compromises neuronal viability. (2) Sense and antisense transcription of the C9ORF72 HRE produces G4C2 and C4G2 transcripts that accumulate in cell nuclei and sequester RNA-binding proteins, resulting in RNA foci. (3) Sense and antisense HRE transcripts are translated via an abnormal mechanism into toxic poly-dipeptides. These aggregation-prone poly-dipeptides have been found in the brains and spinal cords of C9ORF72 ALS and FTD patients and are toxic in cell culture and animal models. Meijboom et al.
  • the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophages.
  • the CRISPR/Cas9 system exploits RNA-guided DNA-binding and sequence-specific cleavage of a target DNA.
  • a guide RNA can be complementary to a target DNA sequence upstream of a PAM (protospacer adjacent motif) site.
  • the Cas (CRISPR-associated) 9 protein binds to the gRNA and the target DNA and introduces a double- strand break (DSB) in a defined location upstream of the PAM site.
  • DSB double- strand break
  • the CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence.
  • Development of epigenome editing tools in manipulating gene expression and/or 3D chromatin structures can help modify an epigenome of cells and treat disorders.
  • SUMMARY The present disclosure provides for a method of treating amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) in a subject.
  • the present disclosure also provides for a method of ameliorating motor neuron degeneration in a subject.
  • the method may comprise administering to the subject a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase)-dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.
  • DNase deoxyribonuclease
  • the method may comprise administering to the subject a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) (or Cpf1 (dCpf1)) and an effector domain, or a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) (or Cpf1 (dCpf1)) and an effector domain; and (b) one or more guide sequences that hybridize to one or more target sequences, or a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.
  • DNase deoxyribonuclease
  • dCas9 or Cpf1 (dCpf1)
  • an effector domain or a first polynucleotide sequence encoding a fusion protein comprising a deoxyribon
  • the method may comprise contacting the cell with a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene.
  • a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene.
  • DNase deoxyribonuclease
  • the method may comprise contacting the cell with a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain, or a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene, or a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene.
  • a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (d
  • the first polynucleotide sequence and the second polynucleotide sequence may be on a single vector or on different vectors.
  • the one or more target sequences are in or near the C9orf72 gene.
  • the one or more target sequences are in GGGGCC (G4C2) repeats in or near the C9orf72 gene and/or a C9orf72 promoter region.
  • the one or more target sequences are in CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene.
  • the cell may be an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).
  • the iPSC may be derived from a fibroblast of a subject (e.g., a subject having ALS or FTD).
  • the method may further comprise culturing the iPSC to differentiate into a neuron.
  • the method may further comprise administering the neuron to a subject (e.g., a subject having ALS or FTD, a subject from whom the iPSC is derived, etc.).
  • a subject e.g., a subject having ALS or FTD, a subject from whom the iPSC is derived, etc.
  • Figure 1A (Upper panel) CRISPR/Cas9 design scheme to delete GGGGCC (G4C2) hexanucleotide repeats in patient-derived induced pluripotent stem cells [iPSCs]. (Lower panel) Double positive (GFP, mCherry) cells collected post- electroporation through fluorescence-activated cell sorting (FACs) in gating E and maintained as single cell colonies for individual verification of G4C2 repeat deletion.
  • Figure 1B Deletion of G4C2 repeats verified through use of repeat-primed PCR as seen in absence of saw-tooth pattern in edited patient-derived iPSC lines.
  • Figure 1C Characterization of G4C2 repeats in both patient- derived iPSCs and control human embryonic stem cells (ESCs) and HEK293 cells.
  • Figure 1D IF (immunofluorescence) staining of C9orf72 in cells described in Figure 1C.
  • Figure 1E Western blot of cells described in Figure 1C.
  • Figure 1F qPCR of C9orf72 transcriptional variants in cells described in Figure 1C.
  • Figure 1G Pyro-seq of the CGI1 in isogenic pairs of C9-ALS iPSCs.
  • Figure 1H Endogenous DPR detection in patient-derived iPSCs.
  • Figure 2A (Upper panel) DNMT3A tethered to CRISPR/dCas9 design scheme to edit DNA methylation along G4C2 repeat region. (Lower panel) Double-positive cells (GFP, mcherry) collected post-transduction through FACs in quadrant C-Q2 and treated with doxycycline to induce methylation.
  • Figure 2B Relative quantitation using RT- qPCR demonstrates efficacy of doxycycline induction of both dCas9 (GFP) and targeting sgRNA (mcherry) for DNA methylation editing.
  • Figure 2C Relative quantitation using RT-qPCR indicates little effect of DNA hypermethylation of G4C2 repeats on C9ORF72 expression level for all three transcripts.
  • Figure 2D Pyrosequencing reflects little change in methylation level at CGI-1.
  • Figure 2E (Left) Representative images of FISH suggest that hypermethylation along G4C2 repeat region has rescue effect by helping to reduce RNA foci in targeted groups. (Right) Quantification of RNA foci.
  • Figure 2F Motor neurons generated from parental and isogenic line respectively double-stained with established motor neuron markers ISL1/2 and HB9 indicate working differentiation protocol for examination of effect of DNA methylation editing in motor neuron context.
  • Figures 3A-3E A C9orf72 BAC, dCas9-DNMT3A mouse model for observing methylation effects on C9-ALS/FTD molecular pathology.
  • Figure 3A- Figure 3B Experimental design for in vivo experiment to determine sex differences in the in vivo, methylation-edited and non-edited contexts.
  • CRISPR/dCas9-DNMT3A in vivo methylation editing
  • sgRNAs targeting CGI-1, CGI-2, and the G4C2 repeat These animals will be divided based on sex and age at harvest to observe younger and older male and female mice.
  • Figure 3C AAV9 transduction in the edited C9-ALS mouse brain.
  • Figure 3D- Figure 3E Reduction in G4C2 foci burden in edited tissue.
  • the present systems/compositions can be used to treat neurodegenerative diseases including motor neuron diseases.
  • the present systems/compositions e.g., the CRISPR/dCas9-Dnmt/Tet system
  • methods may be used to modify the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region in a subject having amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the present systems/compositions and methods may be used to modify neurons (e.g., motor neurons) in vitro or in C9orf72 animal models (e.g., mouse models).
  • Targeted DNA methylation editing of C9orf72 can reduce the formation of RNA foci and production of dipeptides to ameliorate the motor neuron degeneration.
  • the present systems can precisely edit the epigenome, including, but not limited to, DNA methylation, histone acetylation, and DNA looping, at one or multiple genomic loci in mammalian cells, both in vitro and in vivo (e.g., in a patient, in an animal model such as mice, etc.).
  • the system may comprise a catalytically dead Cas9 (dCas9) or Cpf1 (dCpf1), fused with one or more effector protein/domain, including, but not limited to, Dnmt3a, Dnmt3b, Tet1, Tet2, p300, and CTCF, that can modify the state of DNA methylation, histone acetylation, DNA looping, etc.
  • dCas9 catalytically dead Cas9
  • Cpf1 dCpf1
  • the present disclosure provides for a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.
  • DNase deoxyribonuclease
  • dCas9 dead Cas9
  • dCpf1 Cpf1
  • the present disclosure provides for a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain, or a first polynucleotide sequence encoding the fusion protein; and (b) one or more guide sequences that hybridize to one or more target sequences, or a second polynucleotide sequence encoding the one or more guide sequences.
  • the effector domain is TET2, Dnmt3b or CTCF.
  • the effector domain is CTCF where the polypeptide can modify DNA looping.
  • catalytically inactive Cas9 (dCas9) or Cpf1 (dCpf1) is fused with Tet2, Dnmt3b, CTCF, Tet1, Dnmt3a, or p300.
  • targeting of the fusion protein to a hypermethylated or unmethylated/hypomethylated promoter, or enhancer may activate or silence the expression of a gene.
  • Targeted de novo methylation of a CTCF loop anchor site by the fusion protein may block CTCF binding and interfere with DNA looping, which may alter gene expression in the neighboring loop.
  • the target sequence may be located in a non-coding region of a gene.
  • the target sequence may be located in, or near, a promoter, a differentially methylated region (DMR), an enhancer, and/or a CTCF binding site, of a gene.
  • the target sequence may comprise a promoter, a DMR, an enhancer, and/or a CTCF binding site, of a gene.
  • the one or more target sequences (e.g., genomic sequences) may be located within 50 kB of the transcription start site (TSS) of a gene.
  • TSS transcription start site
  • the target sequence may be located in a coding region of a gene.
  • the target sequence may be located in, or near, a promoter, a differentially methylated region (DMR), an enhancer, and/or a CTCF binding site, of a disease-associated (or disease- related) gene.
  • the target sequence may comprise a promoter, a DMR, an enhancer, and/or a CTCF binding site, of a disease-associated gene (disease- related gene).
  • the disease-related gene is C9orf72.
  • the one or more target sequences may be in the C9orf72 gene.
  • the one or more target sequences may be in a non-coding region of the C9orf72 gene.
  • the one or more target sequences may be in a coding region of the C9orf72 gene.
  • the one or more target sequences may be in the GGGGCC (G4C2) repeats in the C9orf72 gene and/or a C9orf72 promoter region.
  • the one or more target sequences may be in CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene.
  • the present system/composition may modify the methylation state of the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region.
  • the present system/composition may modify the methylation state of CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene.
  • the present system/composition may decrease (or increase) the methylation level of the G4C2 repeats in the C9orf72 gene, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8
  • the present system/composition may decrease (or increase) the methylation level of CGI (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6
  • the present system/composition may increase the protein (or transcript) level of C9orf72 by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about
  • the present system/composition may decrease the level/number of RNA foci in the cells by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about
  • the present system/composition may decrease the level of poly-dipeptides in the cells by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about
  • the target sequence may be a genomic sequence.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 target sequences are modified in the cell.
  • the guide sequence may be a CRISPR RNA (crRNA) molecule, a single-guide RNA (sgRNA) molecule, a guide RNA (gRNA), or combinations thereof.
  • the first polynucleotide sequence and the second polynucleotide sequence may be on a single vector, or on different vectors.
  • the second polynucleotide sequence may encode two or more guide sequences that hybridize to two or more target sequences.
  • the system contains an all-in-one vector expressing a chimeric protein (or fusion protein), and one crRNA or an array of crRNAs to target the chimeric protein to one or mulitple genomic loci to mediate epigenome editing.
  • a polynucleotide comprising: (a) a first sequence encoding a fusion protein comprising deoxyribonuclease (DNase) dead (or a catalytically dead) nuclease and an effector domain; and (b) a second sequence encoding one, two or more guide sequences that hybridize to one, two or more genomic sequences.
  • DNase deoxyribonuclease
  • the present disclosure provides for a polynucleotide comprising: (a) a first sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) a second sequence encoding one, two or more guide sequences that hybridize to one, two or more genomic sequences.
  • DNase deoxyribonuclease
  • dCas9 Cpf1
  • the present disclosure provides a pharmaceutical composition comprising: a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein, a system described herein, or a cell(s) described herein.
  • the present disclosure provides a method of modulating an epigenome of a cell.
  • the method may comprise contacting the cell with the present polynucleotide(s) (nucleic acid(s)), present system, or present composition.
  • the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, the method comprising contacting the cell with the present polynucleotide(s) (nucleic acid(s)), present system, or present composition.
  • the present disclosure provides for a method for modifying an epigenome of a cell.
  • the method may comprise contacting the cell with the present system.
  • the methods further comprise introducing the cell into a non- human mammal.
  • the non-human mammal may be a mouse.
  • the disclosure features a method of treating a subject.
  • the method may comprise administering to the subject (or contacting the cell of the subject), an effective amount of the present polynucleotide(s) (nucleic acid(s)), present system, or present composition.
  • the present disclosure provides a method of treating a disease or condition in a subject.
  • the method may comprise administering the present polynucleotide(s) (nucleic acid(s)), present composition, present system, or present cells to the subject.
  • the subject is an animal or plant.
  • the subject is a mammal, primate, or human.
  • the present system/method may be used to treat a neurodegenerative disease such as a motor neuron disease (e.g., ALS).
  • the present system/method may be used to treat a neurodegenerative disease such as frontotemporal dementia (FTD).
  • FTD frontotemporal dementia
  • Cell replacement therapy can be used to prevent, correct or treat diseases, where the methods of the present disclosure are applied to isolated patient’s cells (ex vivo), which is then followed by the injection of “corrected” cells back into the patient.
  • a patient’s iPSC cells may be isolated and differentiated into neurons ex vivo.
  • the patient’s iPSC cells or neurons characterized by the different methylation state of DNA (compared to the healthy subjects) in a disease-related gene may be manipulated using methods of the present disclosure in a manner that results in the modification/correction of the DNA methylation state of a disease-related gene.
  • iPS cells Induced pluripotent stem cells
  • iPS cells refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as a fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.
  • the present methods may further comprise differentiating the iPSC cell to a differentiated cell, for example, a neuron.
  • patient fibroblast cells can be collected from the skin biopsy and transformed into iPSC cells. Dimos JT et al.
  • the cell may be autologous or allogeneic to the subject who is administered the cell.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the same individual.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals of the same species are said to be allogeneic to one another.
  • the corrected cells for cell therapy may be administered to a subject.
  • Cells (e.g., neurons) described in the present disclosure may be formulated with a pharmaceutically acceptable carrier. For example, cells can be administered alone or as a component of a pharmaceutical formulation.
  • the cells can be administered in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents.
  • BSS balanced salt solution
  • the present disclosure provides for a method for modifying a neurodegenerative disease (such as a motor neuron disease) related gene in a cell.
  • the present disclosure provides for a method for modifying an ALS-related gene in a cell.
  • the present disclosure provides for a method for modifying an FTD-related gene in a cell.
  • the method may comprise contacting the cell with the present system, polynucleotide(s) or composition.
  • the cell may be from a subject having a disease, such as a neurodegenerative disease (for example, a motor neuron disease).
  • the cell may be from a subject having ALS or FTD.
  • the cell may be derived from a cell from a subject having a disease, such as a neurodegenerative disease (for example, a motor neuron disease).
  • the cell may be derived from a cell from a subject having ALS or FTD.
  • the cell may be a stem cell, a neuron, a post-mitotic cell, or a fibroblast.
  • the cell is a human cell or a mouse cell.
  • the cell may be an induced pluripotent stem cell (iPSC), e.g., derived from a fibroblast of a subject.
  • the cell may be an ESC.
  • the method may further comprise culturing the iPSC or ESC to differentiate into, e.g., a neuron.
  • the method may further comprise administering the differentiated cell (e.g., a neuron) to a subject.
  • the cell may be autologous or allogeneic to the subject.
  • the present disclosure provides for a method for treating a neurodegenerative disease (including a motor neuron disease) in a patient.
  • the method may comprise administering the present system to the patient.
  • the present polypeptide(s)/system may be used in a method for modifying an epigenome of a cell or a genomic sequence in a cell.
  • the method comprises contacting the cell with the present system/polynucleotide(s).
  • the genomic sequence may be any suitable genomic sequence.
  • the present systems/methods may allow precise gene activation or silencing.
  • the present systems/methods may enable multiplex editing of more than one genomic locus.
  • the present systems/methods can allow epigenome editing at multiple sites using a single vector.
  • U.S. Patent Publication No. 20190359959 is incorporated by reference herein in its entirety.
  • the present disclosure provides for a method for modifying an X-linked disease-related gene or an imprinting-related disease-related gene in a cell.
  • the present systems/methods can be used to treat a disorder/disease.
  • the systems/methods can be applied to reactivate the wild type allele of a gene associated with an X-linked disease, or a gene associated with an imprinting-related disease, via epigenetic editing.
  • one or more of the target sequences are associated with a disease or condition.
  • the present system may target a target sequence that is associated with a disease-related gene, such as a gene associated with a neurodegenerative disease, a gene associated with a motor neuron disease, a gene associated with an X-linked disease, or a gene associated with an imprinting- related disease.
  • the method may further comprise administering to the subject an agent that inhibits or enhances DNA methylation.
  • the agent may be a small molecule.
  • the agent is 5-azacytidine or 5-azadeoxycytidine.
  • the method may further comprise contacting the cell with an agent that inhibits or enhances DNA methylation.
  • the agent may be a small molecule.
  • the agent is 5-azacytidine or 5-azadeoxycytidine.
  • the present disclosure provides for a method of modifying a disease-related gene. The method may comprise introducing the present system/polynucleotide(s) into a cell.
  • the present disclosure provides for gene editing methods that can modify the disease- related gene, which in turn can be used for in vivo gene therapy for patients afflicted with the disease. Furthermore, methods of the present disclosure may be applied to specific gene- humanized mouse model as well as patient-derived cells, allowing for determining the efficiency and efficacy of designed sgRNA and site-specific recombination frequency in human cells, which can be then used as a guide in a clinical setting.
  • the present disclosure provides a cell comprising: a system described herein, a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein; or a composition described herein.
  • the cell may be a vertebrate, mammalian (e.g., human), rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, or primate, cell.
  • the cell may be a plant cell.
  • the cell is a human cell.
  • the cell may be somatic cells, stem cells, mitotic or post-mitotic cells, neurons, fibroblasts, or zygotes.
  • a cell, zygote, embryo, or post-natal mammal can be of vertebrate (e.g., mammalian) origin.
  • the vertebrates are mammals or avians.
  • primate e.g., human
  • rodent e.g., mouse, rat
  • canine feline, bovine, equine, caprine, porcine, or avian (e.g., chickens, ducks, geese, turkeys) cells, zygotes, embryos, or post-natal mammals.
  • the cell, zygote, embryo, or post-natal mammal is isolated (e.g., an isolated cell; an isolated zygote; an isolated embryo).
  • a mouse cell, mouse zygote, mouse embryo, or mouse post-natal mammal is used.
  • a rat cell, rat zygote, rat embryo, or rat post-natal mammal is used.
  • a human cell, human zygote or human embryo is used.
  • the cell may be a somatic cell, germ cell, or prenatal cell.
  • the cell may be a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the present disclosure provides a kit comprising: a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein; a system described herein, or a composition described herein.
  • the kit may comprise an instruction for using the system, the polypeptide(s), the nucleic acid(s), the vector(s), or the composition, in a method described herein.
  • Cas enzymes The Cas enzyme of the CRISPR/Cas system may be Cas9, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, 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, Cpf1, homologs thereof,
  • the Cas enzyme is Cas9.
  • Cas9 is a CRISPR associated endonuclease.
  • Non-limiting exemplary Cas9s are provided herein, e.g. the Cas9 provided for in UniProtKB G3ECR1 (CAS9_STRTR) or the Staphylococcus aureus Cas9, as well as the nuclease dead Cas9, orthologs and biological equivalents each thereof.
  • Orthologs include but are not limited to Streptococcus pyogenes Cas9 (“spCas9”); Cas 9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida; and Cpf1 (which performs cutting functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.
  • spCas9 Streptococcus pyogenes Cas9
  • Cas 9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida
  • Cpf1 which performs cutting functions analogous to Cas9 from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.
  • a nuclease-defective or nuclease-deficient Cas protein (e.g., dCas9) with one or more mutations on its nuclease domains retains DNA binding activity when complexed with a guide sequence (e.g., gRNA).
  • dCas protein can tether and localize effector domains or protein tags by means of protein fusions to sites matched by a guide sequence (e.g., gRNA), thus constituting an RNA-guided DNA binding enzyme.
  • the nucleotide sequence encoding the Cas (e.g., Cas9) nuclease is modified to alter the activity of the protein.
  • the Cas (e.g., Cas9) nuclease is a catalytically inactive Cas (e.g., Cas9) (or a catalytically deactivated/defective Cas9 or dCas9).
  • dCas e.g., dCas9
  • Cas protein e.g., Cas9 that lacks endonuclease activity due to point mutations at one or both endonuclease catalytic sites (RuvC and HNH) of wild type Cas (e.g., Cas9).
  • dCas9 contains mutations of catalytically active residues (D10 and H840) and does not have nuclease activity. In some cases, dCas has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA. As a non-limiting example, in some cases, dCas9 harbors both D10A and H840A mutations of the amino acid sequence of S. pyogenes Cas9.
  • a dCas9 has reduced or defective catalytic activity
  • a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or a A987 mutation (or combinations thereof), e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A(or combinations thereof)
  • the Cas protein can still bind to target DNA in a site-specific manner, because it is still guided to a target polynucleotide sequence by a DNA-targeting sequence of the subject polynucleotide (e.g., gRNA), as long as it retains the ability to interact with the Cas-binding sequence of the subject polynucleotide (e.g., gRNA).
  • the nuclease may be a deoxyribonuclease (DNase) dead (or a catalytically dead) Cas9 (e.g., spCas9).
  • the catalytically dead Cas9 (dCas9) may contain one or more of the following mutations: D10A and H840A.
  • the DNase catalytically dead Cas9 (dCas9) or Cpf1 (dCpf1) has RNase activity.
  • Cpf1 may be from Flavobacterium brachiophilum, Parcubacteria bacterium, Peregrinibacteria bacterium, Acidaminococcus sp., Porphyromonas macacae, Lachnospiraceae bacterium, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida, Candidatus methanoplasma termitum, or Eubacterium eligens.
  • dCpf1 is catalytically dead LbCpf1 (from Lachnospiraceae bacterium).
  • dCpf1 is catalytically dead AsCpf1 (from Acidaminococcus sp.). In yet another embodiment, dCpf1 is catalytically dead FbCpf1 (from Flavobacterium brachiophilum). AsCpf1 may have the UniProt number UniProtKB-U2UMQ6 (CS12A_ACISB), and comprise the corresponding amino acid sequence. LbCpf1 may have the UniProt number UniProtKB-A0A182DWE3 (A0A182DWE3_9FIRM), and comprise the corresponding amino acid sequence.
  • the nuclease may be a deoxyribonuclease (DNase) dead (or a catalytically dead) Cpf1 (dCpf1).
  • the dCpf1 may comprise one or more of the following mutations: D908A, E993A, R1226A and D1263A.
  • the dCpf1 may be Cpf1 comprising the following mutation: D833A.
  • the proteins/polypeptides may also comprise other sequences.
  • an accession number e.g., a UniProt number
  • the accession number refers to one embodiment of the protein or gene which may be used with the systems/methods of the present disclosure.
  • Effector domains The effector domain may have an activity to modify the epigenome of a cell.
  • the effector domain may be a molecule (e.g., protein or a polypeptide) that modulates the expression and/or activation of a genomic sequence (e.g., gene).
  • the effector domain modifies one or both alleles of a gene.
  • the effector domain can be introduced as a nucleic acid sequence and/or as a protein.
  • the effector domain can be a constitutive or an inducible effector domain.
  • a Cas e.g., dCas9, dCpf1, etc.
  • an effector domain nucleic acid sequence are introduced into the cell as a chimeric sequence.
  • the effector domain is fused to a molecule that associates with (e.g., binds to) Cas protein (e.g., the effector molecule is fused to an antibody or antigen binding fragment thereof that binds to Cas protein).
  • a Cas (e.g., dCas9, dCpf1, etc.) protein or variant thereof and an effector domain are fused or tethered creating a chimeric protein and are introduced into the cell as the chimeric protein.
  • the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain bind due to a protein- protein interaction.
  • the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain are covalently linked.
  • the effector domain associates non-covalently with the Cas (e.g., dCas9, dCpf1, etc.) protein.
  • a Cas (e.g., dCas9, dCpf1, etc.) nucleic acid sequence and an effector domain nucleic acid sequence are introduced as separate sequences and/or proteins.
  • the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain are not fused or tethered.
  • fusions of a catalytically inactive Cas protein e.g., dCas9, dCpf1, etc.
  • a catalytically inactive Cas protein e.g., dCas9, dCpf1, etc.
  • all or a portion of (e.g., biologically active portion of) an (one or more) effector domain create chimeric proteins that can be guided to specific DNA sites by one or more guide sequences, to modulate activity and/or expression of one or more genomic sequences (e.g., exert certain effects on transcription or chromatin organization, or bring specific kind of molecules into specific DNA loci, or act as sensor of local histone or DNA state).
  • fusions of dCas9 (or dCpf1) tethered with all or a portion of an effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences to modulate or modify methylation or demethylation of one or more genomic sequences.
  • a "biologically active portion of an effector domain” is a portion that maintains the function (e.g., completely, partially, minimally) of an effector domain (e.g., a "minimal" or "core” domain).
  • the effector domain may be an enzyme that modifies methylation state of DNA.
  • the effector domain may have methylation activity or demethylation activity (e.g., DNA methylation or DNA demethylation activity).
  • the effector domain may be a DNA methyltransferase (DNMT, such as Dnmt3b and Dmnt3a) or a Ten-Eleven-Translocation (TET) methylcytosine dioxygenase protein (such as Tet2 or Tet1).
  • DNMT DNA methyltransferase
  • TET Ten-Eleven-Translocation
  • the effector domain may be ACIDA, MBD4, Apobec1, Apobec2, Apobec3, Tdg, Gadd45a, Gadd45b, or ROS1.
  • the effector domain may be Dnmt1, Dnmt3a, Dnmt3b, CpG Methyltransferase M.SssI, or M.EcoHK3 II.
  • the effector domain may be an enzyme that modifies a histone subunit, such as a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase (e.g., LSD1).
  • HAT histone acetyltransferase
  • HDAC histone deacetylase
  • HMT histone methyltransferase
  • LSD1 histone demethylase
  • the HAT is p300.
  • the effector domain may be CTCF, including wild type CTCF or a DNA binding mutant CTCF.
  • the DNA binding mutant CTCF comprises one or more of the following mutations: K365A, R368A, R396A, and Q418A.
  • the effector domain may be a transcriptional activation domain, such as a transcriptional activation domain derived from VP64, VPR or NF- ⁇ B p65.
  • the effector domain may be a transcriptional silencer (heterochromatin protein 1 (HP1), or Methyl CpG binding Protein 2 (MeCP2)) or transcriptional repression domain (e.g., a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID)).
  • HP1 heterochromatin protein 1
  • Methyl CpG binding Protein 2 Methyl CpG binding Protein 2
  • transcriptional repression domain e.g., a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin
  • effector domains also include a transcription(al) activating domain, a coactivator domain, a transcription factor, a transcriptional pause release factor domain, a negative regulator of transcriptional elongation domain, a transcriptional repressor domain, a chromatin organizer domain, a remodeler domain, a histone modifier domain, a DNA modification domain, and an RNA binding domain.
  • Other examples of effector domains include histone marks readers/interactors and DNA modification readers/interactors.
  • fusion of the dCas9 (or dCpf1) to an effector domain can be to that of a single copy or multiple/tandem copies of full-length or partial-length effector domains.
  • fusions can be with split (functionally complementary) versions of the effector domains.
  • Other examples of effector domains are described in PCT Publication No. WO2014172470 and U.S. Publication No. US20160186208, which are incorporated herein by reference in their entirety.
  • the Cas (e.g., dCas9, dCpf1, etc.) protein can be fused to the N-terminus or C-terminus of the effector domain.
  • fusion of dCas9 (or dCpf1) with all or a portion of one or more effector domains comprise one or more linkers.
  • a linker comprises one or more amino acids.
  • a linker comprises two or more amino acids.
  • a linker comprises the amino acid sequence GS.
  • fusion of Cas e.g., dCas9, dCpf1, etc.
  • two or more effector domains comprises one or more interspersed linkers (e.g., GS linkers) between the domains.
  • one or more nuclear localization sequences may be located between the catalytically inactive nuclease (e.g., dCas9, dCpf1, etc.) and the effector domain.
  • a target sequence is located within or near an essential gene or a non-essential gene.
  • the target sequence may be derived from a gene (e.g., a disease-related gene) described herein.
  • one copy of one or more genomic sequences in the cell is modified.
  • both copies of one or more of the genomic sequences in the cell are modified.
  • the one or more genomic sequences that are modified are endogenous to the cell.
  • at least two of the genomic sequences are endogenous genomic sequences.
  • at least two of the genomic sequences are exogenous genomic sequences.
  • at least one of the genomic sequences is an endogenous genomic sequence and at least one of the genomic sequences is an exogenous genomic sequence.
  • the genomic sequences are endogenous genes. In some aspects, at least two of the genomic sequences are exogenous genes. In some aspects where there are at least two genomic sequences, at least one of the genomic sequences is an endogenous gene and at least one of the genomic sequences is an exogenous gene. In some aspects, at least two of the genomic sequences are at least 1 kB apart. In some aspects, at least two of the genomic sequences are on different chromosomes.
  • the target sequence may comprise a differentially methylated region (DMR).
  • DMR differentially methylated region
  • a differentially methylated region may be differentially methylated between cells of different cell types (e.g., muscle cells vs neuron, or skin cells vs hepatocytes).
  • a differentially methylated region may be differentially methylated between diseased vs non-diseased cells (e.g., cancer vs non- cancer cells).
  • a differentially methylated region may be differentially methylated between differentiation states (e.g., progenitor cells vs terminally differentiated cells). The effect on expression of one or more genes (e.g., within up to about 0.5, 1, 2, 5, 10, 20, 50, 100, 500 kb or within about 1, 2, 5, or 10 MB from the modification) may be assessed.
  • the differentially methylated region may be hypermethylated, unmethylated or hypomethylated.
  • a differentially methylated region is located within 50 kB of the transcription start site of the gene.
  • the method may comprise contacting the cell with the present system, where the guide sequence targets the differentially methylated region.
  • the differentially methylated region is hypermethylated in the cell and the effector domain (e.g., Tet2 or Tet1) has demethylation activity.
  • the differentially methylated region is unmethylated or hypomethylated in the cell and the effector domain (e.g., Dnmt3a, Dnmt3b) has methylation activity.
  • the present system/method may demethylate a genomic sequence that is aberrantly hypermethylated, or may methylate a genomic sequence that is aberrantly unmethylated or hypomethylated.
  • an aberrantly hypermethylated sequence, or aberrantly unmethylated or hypomethylated sequence may occur in a disease or disorder.
  • Modifying the methylation or demethylation of the CTCF site may treat or prevent a disease or disorder that exhibits an aberrantly unmethylated or hypomethylated sequence or region, or an aberrantly hypermethylated sequence or region.
  • a CTCF loop may be opened by methylating a CTCF binding site and thereby bring a gene that is outside the loop under control of an enhancer inside the loop if one wanted to increase expression of that gene (e.g., if expression of the gene is aberrantly low and/or if increased expression is desired for therapeutic or other purposes).
  • the present system/method may modify a promoter sequence.
  • Targeting of the present system to methylated, hypermethylated, unmethylated or hypomethylated promoter sequences may cause activation or silencing of expression of a gene.
  • the present system/method may modify an enhancer sequence. Targeting of the present system to methylated, hypermethylated, unmethylated or hypomethylated enhancer sequences may cause activation or silencing of expression of a gene.
  • the present system/method may modify a CTCF binding site. Targeting of the present system to CTCF binding sites may affect CTCF binding and interfere with, or increase, DNA looping, which may alter gene expression (e.g., in the neighboring loop).
  • multiple genomic sequences are modulated (e.g., multiplexed activation).
  • All or a portion of the region targeted by the one or more guide sequences may be a differentially methylated region.
  • the differentially methylated region is exactly or within about 25 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1500 bases, 2000 bases, 5000 bases, 10000 bases, 20000 bases, 50000 bases or more upstream to the one or more genes (e.g., endogenous genes; exogenous genes) or a (one or more) transcription start site (TSS).
  • genes e.g., endogenous genes; exogenous genes
  • TSS transcription start site
  • the differentially methylated region is exactly or within about 25 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1500 bases, 2000 bases, 5000 bases, 10000 bases, 20000 bases, 50000 bases, or more downstream to the one or more genes (e.g., endogenous genes; exogenous genes) or a TSS.
  • the regulatory region targeted by one or more guide sequences may be entirely or partially found at or about the 5’ end of the gene (e.g., endogenous or exogenous) or a TSS.
  • the 5’ end of a gene can include un- transcribed (flanking) regions (e.g., all or a portion of a promoter) and a portion of the transcribed region.
  • "Modulate” or “modify” means to cause or facilitate a qualitative and/or quantitative change, alteration, or modification in a level (expression level), an activity, a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon.
  • the present system/method may result in an increase of the expression level or activity of at least one (wildtype) gene or protein, or a decrease of the expression level or activity of at least one (mutant) gene or protein, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99%, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days,
  • the expression level and/or activity of the (wildtype) gene or protein may increase, or the expression level and/or activity of the (mutant) gene or protein may decrease, by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7
  • the gRNA may contain a targeting segment that can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target sequence ("target region" or "target DNA").
  • a targeting segment that can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
  • the gRNA (or crRNA, or sgRNA) sequence (or the targeting segment of the gRNA (or crRNA, or sgRNA)) has 100% complementarity to the target sequence.
  • the targeting segment of the gRNA (or crRNA, or sgRNA) may have full complementarity with the target sequence.
  • the targeting segment of the gRNA (or crRNA, or sgRNA) may have partial complementarity with the target sequence.
  • the targeting segment of the gRNA (or crRNA, or sgRNA) has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the target sequence (mismatches).
  • the gRNA (or crRNA, or sgRNA) is about 10 nucleotides to about 150 nucleotides in length.
  • the targeting segment of the gRNA (or crRNA, or sgRNA) is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides in length.
  • the targeting segment of the gRNA (or crRNA, or sgRNA) is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.
  • the targeting segment of the gRNA is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
  • the degree of complementarity, together with other properties of the gRNA (or crRNA, or sgRNA) is sufficient to allow targeting of a Cas molecule to the target nucleic acid.
  • gRNA or “guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique. Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art.
  • gRNA may comprise, or alternatively consist essentially of, or yet further consist of, a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).
  • a gRNA is synthetic (Kelley, M.
  • a biological equivalent of a gRNA includes but is not limited to polynucleotides or targeting molecules that can guide a Cas or equivalent thereof to a specific nucleotide sequence such as a specific region of a cell’s genome.
  • the guide sequence (e.g., crRNA, sgRNA, gRNA, etc.) used in the present system/method can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 5960, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 9192, 93, 94, 95, 96, 97, 98, 99, or 100 nucle
  • the guide sequence (e.g., crRNA, sgRNA, gRNA, etc.) can be between about 15 and about 30 nucleotides in length (e.g., about 15-29, 15-26, 15-25; 16-30, 16-29, 16-26, 16-25; or about 18-30, 18-29, 18-26, or 18-25 nucleotides in length).
  • the one or more guide sequences also comprise a (one or more) binding site for a (one or more) catalytically inactive site-specific nuclease.
  • the catalytically inactive site-specific nuclease may be a catalytically inactive CRISPR associated (Cas) protein, such as dCas9, and dCpf1.
  • Cas CRISPR associated
  • the catalytically inactive site-specific nuclease binds to the one or more guide sequences.
  • the guide sequence is an RNA sequence.
  • a single RNA sequence can be complementary to one or more (e.g., all) of the genomic sequences that are being modulated or modified.
  • a single RNA is complementary to a single target genomic sequence.
  • RNA sequences are used wherein each RNA sequence is complementary to (specific for) one target genomic sequence.
  • two or more, three or more, four or more, five or more, or six or more RNA sequences are complementary to (specific for) different parts of the same target sequence.
  • two or more RNA sequences bind to different sequences of the same region of DNA.
  • a single RNA sequence is complementary to at least two target or more (e.g., all) of the genomic sequences.
  • the portion of the RNA sequence that is complementary to one or more of the genomic sequences and the portion of the RNA sequence that binds to the catalytically inactive site-specific nuclease can be introduced as a single sequence or as 2 (or more) separate sequences into a cell, zygote, embryo or nonhuman animal.
  • the sequence that binds to the catalytically inactive site-specific nuclease comprises a stem-loop.
  • the system contains one or more guide sequences (or a polynucleotide sequence encoding one or more guide sequences) that are complementary to all or a portion of a (one or more) regulatory region, an open reading frame (ORF; a splicing factor), an intronic sequence, a chromosomal region (e.g., telomere, centromere) of the one or more genomic sequences in a cell.
  • the regulatory region targeted by one or more genomic sequences is a promoter, enhancer, and/or operator region. In some aspects, all or a portion of the regulatory region is targeted by the one or more guide sequences.
  • gRNAs can be generated to target a specific gene, optionally a gene associated with a disease, disorder, or condition.
  • the guide RNAs facilitate the target specificity of the CRISPR/Cas system.
  • Further aspects such as promoter choice, as discussed herein, may provide additional mechanisms of achieving target specificity – e.g., selecting a promoter for the guide RNA encoding polynucleotide that facilitates expression in a particular organ or tissue. Accordingly, the selection of suitable gRNAs for the particular disease, disorder, or condition is contemplated herein.
  • a neurodegenerative disease such as a motor neuron disease
  • Motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome.
  • the method may comprise administering to the subject a therapeutically effective amount of the present system, polynucleotide(s) or composition.
  • Disease disease
  • disorder or “condition” are used interchangeably and may refer to any alteration from a state of health and/or normal functioning of an organism, e.g., an abnormality of the body or mind that causes pain, discomfort, dysfunction, distress, degeneration, or death to the individual afflicted.
  • a disease is a psychiatric, neurological, neurodevelopmental disease, neurodegenerative disease, cardiovascular disease, autoimmune disease, cancer, metabolic disease, or respiratory disease.
  • a disease is a psychiatric, neurological, or neurodevelopmental disease, e.g., schizophrenia, depression, bipolar disorder, epilepsy, autism, addiction.
  • Neurodegenerative diseases include, e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD).
  • a disease exhibits hypermethylation (e.g., aberrant hypermethylation) or unmethylation/hypomethylation (e.g., aberrant unmethylation or hypomethylation) in a genomic sequence.
  • neurodegenerative disease generally refers to any disease, disorder, and/or condition that affects the neurons (sometimes referred to as “nerve cells”), such as neurons of a brain and/or neurons of a nervous system which is associated with the degeneration or loss of neural cells.
  • neurons sometimes referred to as “nerve cells”
  • Neurodegenerative diseases may result in progressive degeneration and/or death of nerve cells.
  • neurodegeneration is the progressive loss of structure and/or function of neurons, including the death of neurons.
  • Neurodegenerative diseases may cause problems with movement (e.g., ataxias), or mental or cognitive functioning (e.g., dementias). Frequently neurodegeneration is associated with neuroinflammation. Therefore, it is to be understood that neurodegenerative diseases or disorders encompass neural diseases which are characterized by neuroinflammation. Sometimes in such diseases activated microglia may produce inflammatory cytokines that contribute to widespread inflammation and may lead to and/or result in a neurodegenerative condition and/or disease. Some neurodegenerative diseases and/or conditions are associated with microglia cell over-activation, increased numbers of microglia cells, production of inflammatory proteins and/or inflammatory activities, and/or neuronal death.
  • the present method/system may decrease or prevent at least one symptom associated with a neurodegenerative disease.
  • the present system/composition and method may result in a decrease in neurodegeneration, degeneration of neurons (e.g., degeneration of motor neurons), the loss of neurons, neuronal cell death, morphological abnormalities of the neuromuscular junctions (NMJs), etc. of the subject, where neurodegeneration, degeneration of neurons, the loss of neurons, neuronal cell death, morphological abnormalities of the neuromuscular junctions (NMJs), etc.
  • the present system/composition and method is no greater than 90%, no greater than 85%, no greater than 80%, no greater than 75%, no greater than 70%, no greater than 65%, no greater than 60%, no greater than 55%, no greater than 50%, no greater than 45%, no greater than 40%, no greater than 35%, no greater than 30%, no greater than 25%, no greater than 20%, no greater than 15%, no greater than 10%, about 10% to about 90%, about 15% to about 80%, about 20% to about 70%, about 25% to about 60%, about 30% to about 50%, about 30% to about 40%, about 25% to about 40%, about 20% to about 30%, about 25% to about 35%, about 10% to about 30%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 20% to about 50%, about 12.5% to about 80%, about 20% to about 70%, about
  • the present method and system/composition may ameliorate the symptoms of a neurodegenerative disease or disorder in a subject.
  • the present method and system/composition may result in at least partial correction of neuropathology, and/or alleviation and/or prevention and/or stabilization and/or slowing of disease progression, and/or progression of the symptoms of a neurodegenerative disease or disorder.
  • the present method and composition may prevent neuron death, and/or delay the onset of paralysis and death.
  • the present system/composition and method may result in an increase in motor neuron number, neuromuscular junction (NMJ) electrophysiology (e.g., miniature end-plate potentials (mEPPs), end-plate potentials (EPPs), Quantal content), neurotransmission at the NMJ, muscle strength, etc. of the subject, where the motor neuron number, neuromuscular junction (NMJ) electrophysiology (e.g., miniature end-plate potentials (mEPPs), end-plate potentials (EPPs), Quantal content), neurotransmission at the NMJ, muscle strength, etc.
  • NMJ neuromuscular junction electrophysiology
  • mEPPs miniature end-plate potentials
  • EPPs end-plate potentials
  • Quantal content neurotransmission at the NMJ, muscle strength, etc.
  • composition and method is at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 1.1-fold, at least or about 1.2-fold, at least or about 1.3-fold, at least or about 1.4-fold, at least or about 1.5-fold, at least or about 1.6-fold, at least or about 1.8-fold, at least or about 15-fold, at least or about 20-fold, at least or about 50-fold, at least or about 100-fold, at least or about 120-fold, from about 2-fold to about 500-fold, from about 1.1-fold to about 10-fold, from about 1.1-fold to about 5-fold, from about 1.5-fold to about 5-fold, from about 2-fold to about 5-fold, from about 3-fold to about 4- fold, from about 5-fold to about 10-fold, from about 5-fold to about 200
  • the methods of the present disclosure may be used to treat patients at a different stage of the disease (e.g., early, middle or late).
  • the present methods may be used to treat a patient once or multiple times.
  • the length of treatment may vary and may include multiple treatments.
  • Delivering the present systems The nuclease (e.g., dCas9, dCpf1, etc.) can be introduced into the cell in the form of DNA, mRNA or protein.
  • the sequence-specific nuclease can be introduced into the cell in the form of a protein or in the form of a nucleic acid encoding the sequence-specific nuclease, such as an mRNA or a cDNA.
  • Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics.
  • CRISPR/Cas may be encoded by a viral vector.
  • the polynucleotide/vector may be a recombinant lentiviral vector, or an adeno-associated viral (AAV) vector, such as an AAV2 vector, or an AAV8 vector.
  • a variety of viral constructs may be used to deliver the present system to the targeted cells and/or a subject.
  • Non-limiting examples of such recombinant viruses include recombinant lentiviruses, recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids, and phages.
  • Options for gene delivery viral constructs are well known (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1):33-40; and Walther W.
  • AAV viral vectors may be selected from among any AAV serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or other known and unknown AAV serotypes.
  • AAV2 and/or AAV8 are used.
  • the term AAV covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome of a second serotype.
  • delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used as an alternative to viral vectors.
  • delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics.
  • RNP ribonucleoprotein
  • lipid-based delivery system lipid-based delivery system
  • gene gun hydrodynamic, electroporation or nucleofection microinjection
  • biolistics biolistics.
  • Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1;459(1-2):70-83).
  • Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue- specific, or species specific.
  • a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, kozak sequences and introns).
  • promoter/regulatory sequences useful for driving constitutive expression of a gene include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like.
  • CMV cytomegalovirus promoter
  • EF1a human elongation factor 1 alpha promoter
  • SV40 simi
  • tissue-specific or inducible promoter/regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer/promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others.
  • promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention.
  • promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention.
  • the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.
  • Vectors according to the present disclosure can be transformed, transfected or otherwise introduced into a wide variety of host cells.
  • Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome.
  • transduction generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.
  • the present system may be delivered by any suitable means. In certain embodiments, the system is delivered in vivo.
  • the system is delivered to isolated/cultured cells (e.g., iPSC cells) in vitro to provide modified cells useful for in vivo delivery to a subject/patient.
  • the disclosure provides for introducing the present system or composition into a eukaryotic cell.
  • the cell may be a stem cell.
  • stem cells include pluripotent, totipotent, multipotent and unipotent stem cells.
  • pluripotent stem cells include embryonic stem cells, embryonic germ cells, fetal stem cells, adult stem cells, embryonic carcinoma cells and induced pluripotent stem cells (iPSCs).
  • the cell may be a somatic cell.
  • Somatic cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line capable of prolonged proliferation in culture (e.g., for longer than 3 months) or indefinite proliferation (immortalized cells).
  • Adult somatic cells may be obtained from individuals, e.g., human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art.
  • Somatic cells of use in aspects of the invention include mammalian cells, such as, for example, human cells, non-human primate cells, or rodent (e.g., mouse, rat) cells.
  • organs e.g., skin, lung, pancreas, liver, stomach, intestine, heart, breast, reproductive organs, muscle, blood, bladder, kidney, urethra and other urinary organs, etc., generally from any organ or tissue containing live somatic cells.
  • Mammalian somatic cells useful in various embodiments include, for example, fibroblasts, Sertoli cells, granulosa cells, neurons, pancreatic cells, epidermal cells, epithelial cells, endothelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc.
  • the present system or composition may be introduced into a cell, a zygote, an embryo, a human subject, or a non-human mammal.
  • the cell is a cancer cell or other cell characterized by a disease or disorder.
  • the target sequence is derived from the nucleic acid of a human cell.
  • the target sequence is derived from the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.
  • the cell is a cell characterized by an unwanted genomic component (e.g., a viral genomic component), such as a cell infected with viruses, a cell infected with bacteria etc.
  • an unwanted genomic component e.g., a viral genomic component
  • Subjects, which may be treated according to the present disclosure include all animals which may benefit from the present invention. Such subjects include mammals, preferably humans (infants, children, adolescents and/or adults), but can also be an animal such as dogs and cats, farm animals such as cows, pigs, sheep, horses, goats and the like, and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
  • the present disclosure provides a pharmaceutical composition comprising the present system.
  • the pharmaceutical composition may contain a pharmaceutically and/or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.
  • the carrier is an isotonic sodium chloride solution.
  • the carrier is balanced salt solution.
  • the carrier includes Tween.
  • viruses may be frozen in the presence of glycerol or Tween-20.
  • the present system, cells or compositions may be administered by, direct delivery to a desired organ or tissue, injection, oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Additionally, routes of administration may be combined, if desired.
  • Administration may be through any suitable routes, including but not limited to, intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, and inhalation.
  • routes including but not limited to, intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, and inhalation.
  • treating or “treatment” of a disease or a condition in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • treatment excludes prevention.
  • Example 1 We hypothesize that the methylation of the C9orf72 locus represents a critical factor in determining the onset and progression of signs and symptoms of ALS in C9orf72 mutant carriers.
  • We observed RNA foci was abolished in G4C2 repeat deletion line and the neuronal activity was elevated in motor neurons derived from this line.
  • GR poly glycine–arginine dipeptide
  • RNA FISH RNA FISH to examine RNA foci containing G4C2 repeat.
  • Figure 1I we detected 6% of patient iPSCs with more than two RNA foci, whereas the isogenic control without G4C2 repeats only show 1% of foci-positive cells.
  • These characterizations of C9orf72-ALS/FTD iPSCs and isogenic controls lay down a solid base to perform DNA methylation editing and evaluate the functional consequence of DNA methylation to neurodegeneration.
  • FIG. 2A we used lentiviral vectors to express dCas9- Dnmt3a-P2A-GFP and sgRNA-mCherry in C9orf72-ALS/FTD iPSC line #52 with about 800 G4C2 repeats and isolated the infection-positive (GFP+;mCherry+) population by FACS.
  • qPCR analysis of three transcriptional variants as well as the total C9orf72 transcripts showed an increase after targeted methylation of G4C2 repeats ( Figure 2B and 2C).
  • Targeted DNA methylation of G4C2 repeats increases the expression of C9orf72 transcription.
  • targeted DNA methylation of the G4C2 repeats reduced the formation of RNA foci, one of the molecular hallmarks for C9-ALS/FTD pathology.
  • Example 2 As illustrated in Figures 3A and 3B, we carried out studies to demonstrate that our in vivo editing model can help ameliorate C9-ALS/FTD molecular phenotypes.
  • mice expressing a BAC carrying the human C9orf72 gene with the hexanucleotide repeat expansion (HRE) with mice carrying the CRISPR/dCas9-DNMT3A-P2A-GFP tool with a GFP reporter.
  • HRE hexanucleotide repeat expansion
  • mTeSR1 medium STMCELL, #85850
  • MEFs mouse embryonic fibroblasts
  • standard hESCs medium [DMEM/F12 (Invitrogen) supplemented with 15% fetal bovine serum (GIBCO HI FBS, 10082-147), 5% KnockOut Serum Replacement (Invitrogen), 2 mM L-glutamine (MPBio), 1% nonessential amino acids (Invitrogen), 1% penicillin-streptomycin (Lonza), 0.1 mM b-mercaptoethanol (Sigma) and 4 ng/ml FGF2 (R&D systems)].
  • iPSCs and neurons were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature.
  • Cells were permeabilized with PBST (1 x PBS solution with 0.1% Triton X-100) before blocking with 10% Normal Donkey Serum (NDS) in PBST.
  • PBST permeabilized with PBST (1 x PBS solution with 0.1% Triton X-100
  • NDS Normal Donkey Serum
  • Cells were then incubated with appropriately diluted primary antibodies in PBST with 5% NDS for 1 hours at room temperature or 12 hours at 4°C, washed with PBST for 3 times at room temperature and then incubated with desired secondary antibodies in TBST with 5% NDS and DAPI to counter stain the nuclei.
  • FACS analysis To isolate the infection-positive cell after lentiviral transduction, the treated cells were dissociated with trypsin and single-cell suspensions were prepared in growth medium subject to a BD FACSAria cell sorter according to the manufacture’s protocol. Data were analyzed with FlowJo software. Western blot Cells were lysed by RIPA buffer with proteinase inhibitor (Invitrogen), and subject to standard immunoblotting analysis. Mouse anti-Cas9 (1:1000, Active Motif), mouse a-Tubulin (1:1000, Sigma), mouse anti-FMR1polyG (1:1000, EMD Millipore), rabbit anti-FMRP (1:100, Cell Signaling) antibodies were used.
  • RT-qPCR Cells were harvested using Trizol followed by Direct-zol (Zymo Research), according to manufacturer’s instructions.
  • RNA was converted to cDNA using First-strand cDNA synthesis (Invitrogen SuperScript III). Quantitative PCR reactions were prepared with SYBR Green (Invitrogen), and performed in 7900HT Fast ABI instrument. Bisulfite Conversion, PCR and Sequencing Bisulfite conversion of DNA was established using the EpiTect Bisulfite Kit (QIAGEN) following the manufacturer’s instructions.
  • the resulting modified DNA was amplified by first round of nested PCR, following a second round using loci specific PCR primers.
  • the first round of nested PCR was done as follows: 94°C for 4 min; 55°C for 2 min; 72°C for 2 min; Repeat steps 1-31 X; 94°C for 1 min; 55°C for 2 min; 72°C for 2 min; Repeat steps 5-735X; 72°C for 5 min; Hold 12°C.
  • the second round of PCR was as follows: 95°C for 4 min; 94°C for 1 min; 55°C for 2 min; 72°C for 2 min; Repeat steps 2-435 X; 72°C for 5 min; Hold 12°C.
  • the resulting amplified products were gel-purified, sub-cloned into a pCR2.1-TOPO-TA cloning vector (Life technologies), and sequenced.

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Abstract

The present disclosure provides for systems and methods for treating neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). For example, a subject having ALS or FTD may be administered a system comprising a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase)-dead CRISPR-Cas nuclease and an effector domain; and a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.

Description

COMPOSITIONS AND METHODS FOR TREATING MOTOR NEURON DISEASES CROSS REFERENCE TO RELATED APPLICATION The present application claims priority to U.S. Provisional Patent Application No. 63/445,902, filed on February 15, 2023, which is hereby incorporated by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under NS126185 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION The present disclosure relates to systems and methods to treat neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). BACKGROUND OF THE DISCLOSURE Genetic studies of epigenetic modifiers such as DNA methyltransferases and histone acetyltransferases have revealed a critical role for epigenetic regulation during development and function. Alteration of epigenetic modifications have been documented in a variety of disorders, including neurological disorders (such as neurodevelopmental, psychiatric, and neurodegenerative diseases), cancer and cardiovascular diseases. Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease in which continuous degeneration of motor neurons eventually causes patient death without effective treatment. The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) mutation in the noncoding region of the Chromosome 9 Open Reading Frame 72 (C9ORF72) gene is the most common genetic cause of ALS. In healthy individuals, the size of the hexanucleotide sequence is less than 24 repeats, but affected individuals have expansion mutations that can number in the thousands of repeats. Meijboom et al. CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Nat. Commun.13, 6286 (2022). However, not all C9orf72 mutant carriers develop ALS and a significant portion of mutant carriers are asymptomatic, suggesting that other factors modify the disease onset such as DNA methylation. Interestingly, hypermethylation of the G4C2 repeats and the C9orf72 promoter region were observed in C9orf72 mutant carriers. The HRE is also the most common genetic cause of frontotemporal dementia (FTD). Both ALS and FTD are aggressive diseases with no treatments that significantly slow disease progression or extend life expectancy. C9ORF72 interacts with endosomes and is required for normal vesicle trafficking, autophagy induction and lysosomal biogenesis in diverse cell types, including motor neurons. The HRE gives rise to three pathological hallmarks of C9ORF72 ALS: (1) It impairs expression, leading to C9ORF72 haploinsufficiency that compromises neuronal viability. (2) Sense and antisense transcription of the C9ORF72 HRE produces G4C2 and C4G2 transcripts that accumulate in cell nuclei and sequester RNA-binding proteins, resulting in RNA foci. (3) Sense and antisense HRE transcripts are translated via an abnormal mechanism into toxic poly-dipeptides. These aggregation-prone poly-dipeptides have been found in the brains and spinal cords of C9ORF72 ALS and FTD patients and are toxic in cell culture and animal models. Meijboom et al. CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Nat. Commun.13, 6286 (2022). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophages. The CRISPR/Cas9 system exploits RNA-guided DNA-binding and sequence-specific cleavage of a target DNA. A guide RNA (gRNA) can be complementary to a target DNA sequence upstream of a PAM (protospacer adjacent motif) site. The Cas (CRISPR-associated) 9 protein binds to the gRNA and the target DNA and introduces a double- strand break (DSB) in a defined location upstream of the PAM site. Geurts et al., Science 325, 433 (2009); Mashimo et al., PLoS ONE 5, e8870 (2010); Carbery et al., Genetics 186, 451-459 (2010); Tesson et al., Nat. Biotech. 29, 695-696 (2011). Wiedenheft et al. Nature 482,331-338 (2012); Jinek et al. Science 337,816-821 (2012); Mali et al. Science 339,823-826 (2013); Cong et al. Science 339,819-823 (2013). The ability of the CRISPR/Cas9 system to be programed to cleave not only viral DNA but also other genes opened a new venue for genome engineering. The CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence. Development of epigenome editing tools in manipulating gene expression and/or 3D chromatin structures can help modify an epigenome of cells and treat disorders. SUMMARY The present disclosure provides for a method of treating amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) in a subject. The present disclosure also provides for a method of ameliorating motor neuron degeneration in a subject. In certain embodiments, the method may comprise administering to the subject a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase)-dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences. In certain embodiments, the method may comprise administering to the subject a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) (or Cpf1 (dCpf1)) and an effector domain, or a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) (or Cpf1 (dCpf1)) and an effector domain; and (b) one or more guide sequences that hybridize to one or more target sequences, or a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences. Also encompassed by the present disclosure is a method for modifying an epigenome of a cell. In certain embodiments, the method may comprise contacting the cell with a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene. In certain embodiments, the method may comprise contacting the cell with a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain, or a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene, or a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in or near the C9orf72 gene. In certain embodiments, the DNase-dead CRISPR-Cas nuclease may be dCas9 or dCpf1. In certain embodiments, the effector domain is a DNA methyltransferase (DNMT) or a Ten- Eleven-Translocation (TET) methylcytosine dioxygenase protein. For example, the TET protein may be Tet1. The DNMT protein may be Dnmt3a. In certain embodiments, the effector domain is TET1 or Dnmt3a. The effector domain may have an activity to modify an epigenome. The effector domain may be an enzyme that modifies the methylation state of DNA. The first polynucleotide sequence and the second polynucleotide sequence may be on a single vector or on different vectors. In certain embodiments, the one or more target sequences are in or near the C9orf72 gene. In certain embodiments, the one or more target sequences are in GGGGCC (G4C2) repeats in or near the C9orf72 gene and/or a C9orf72 promoter region. In certain embodiments, the one or more target sequences are in CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene. The system may modify the methylation state of the G4C2 repeats in or near the C9orf72 gene and/or the C9orf72 promoter region. The system may modify the methylation state of CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene. In certain embodiments, the one or more guide sequences is/are one or more CRISPR RNA (crRNA) molecules, one or more single-guide RNA (sgRNA) molecules, one or more guide RNA (gRNA) molecules, or combinations thereof. In certain embodiments, the second polynucleotide sequence encodes two or more crRNA molecules that hybridize to two or more target sequences. The cell may be a neuron. The cell may be an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC). The iPSC may be derived from a fibroblast of a subject (e.g., a subject having ALS or FTD). The method may further comprise culturing the iPSC to differentiate into a neuron. The method may further comprise administering the neuron to a subject (e.g., a subject having ALS or FTD, a subject from whom the iPSC is derived, etc.). BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1I: Characterization of molecular phenotypes in parental and isogenic C9ORF72-ALS/FTD patient derived iPSCs. Figure 1A: (Upper panel) CRISPR/Cas9 design scheme to delete GGGGCC (G4C2) hexanucleotide repeats in patient-derived induced pluripotent stem cells [iPSCs]. (Lower panel) Double positive (GFP, mCherry) cells collected post- electroporation through fluorescence-activated cell sorting (FACs) in gating E and maintained as single cell colonies for individual verification of G4C2 repeat deletion. Figure 1B: Deletion of G4C2 repeats verified through use of repeat-primed PCR as seen in absence of saw-tooth pattern in edited patient-derived iPSC lines. Figure 1C: Characterization of G4C2 repeats in both patient- derived iPSCs and control human embryonic stem cells (ESCs) and HEK293 cells. Figure 1D: IF (immunofluorescence) staining of C9orf72 in cells described in Figure 1C. Figure 1E: Western blot of cells described in Figure 1C. Figure 1F: qPCR of C9orf72 transcriptional variants in cells described in Figure 1C. Figure 1G: Pyro-seq of the CGI1 in isogenic pairs of C9-ALS iPSCs. Figure 1H: Endogenous DPR detection in patient-derived iPSCs. (Left-most) Representative immunofluorescence images of C9ORF72 haploinsufficiency in patient-derived iPSCs. (Upper middle) Representative immunoblot images of C9ORF72 haploinsufficiency in patient-derived iPSCs. (Lower middle) Relative quantitation of C9ORF72 haploinsufficiency linked to presence of G4C2 repeats by comparing parental and isogenic lines. (Right-most) CpG-island 1 (CGI-1) in promoter region of C9ORF72 gene hypermethylated in parental lines relative to isogenic lines. Figure 1H: DPR OE in HEK293. (Left) No endogenous DPRs detected in patient-derived iPSCs through immunostaining. (Right) Functionality of dipeptide repeat (DPR) detection system checked through DPR over-expression (OE) in HEK293 cells. Figure 1I: (Left) Representative fluorescence in-situ hybridization (FISH) images indicate correlation between RNA foci and presence of G4C2 repeats. (Right) Quantification of RNA foci in both experimental and control groups. Figures 2A-2F: DNA methylation editing using CRISPR/dCas9 system in patient-derived iPSCs and generation of motor neurons. Figure 2A: (Upper panel) DNMT3A tethered to CRISPR/dCas9 design scheme to edit DNA methylation along G4C2 repeat region. (Lower panel) Double-positive cells (GFP, mcherry) collected post-transduction through FACs in quadrant C-Q2 and treated with doxycycline to induce methylation. Figure 2B: Relative quantitation using RT- qPCR demonstrates efficacy of doxycycline induction of both dCas9 (GFP) and targeting sgRNA (mcherry) for DNA methylation editing. Figure 2C: Relative quantitation using RT-qPCR indicates little effect of DNA hypermethylation of G4C2 repeats on C9ORF72 expression level for all three transcripts. Figure 2D: Pyrosequencing reflects little change in methylation level at CGI-1. Figure 2E: (Left) Representative images of FISH suggest that hypermethylation along G4C2 repeat region has rescue effect by helping to reduce RNA foci in targeted groups. (Right) Quantification of RNA foci. Figure 2F: Motor neurons generated from parental and isogenic line respectively double-stained with established motor neuron markers ISL1/2 and HB9 indicate working differentiation protocol for examination of effect of DNA methylation editing in motor neuron context. Figures 3A-3E: A C9orf72 BAC, dCas9-DNMT3A mouse model for observing methylation effects on C9-ALS/FTD molecular pathology. Figure 3A-Figure 3B: Experimental design for in vivo experiment to determine sex differences in the in vivo, methylation-edited and non-edited contexts. We will inject neonates carrying both the human C9orf72 BAC and our in vivo methylation editing (CRISPR/dCas9-DNMT3A) tool with sgRNAs targeting CGI-1, CGI-2, and the G4C2 repeat. These animals will be divided based on sex and age at harvest to observe younger and older male and female mice. We will perform assays to observe RNA foci and dipeptide repeat protein burden along with other cell viability and C9-ALS/FTD hallmarks. We will also perform snRNA-seq on tissue from these samples. Figure 3C: AAV9 transduction in the edited C9-ALS mouse brain. Figure 3D-Figure 3E: Reduction in G4C2 foci burden in edited tissue.
DETAILED DESCRIPTION The present systems/compositions can be used to treat neurodegenerative diseases including motor neuron diseases. For example, the present systems/compositions (e.g., the CRISPR/dCas9-Dnmt/Tet system) and methods may be used to modify the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region in a subject having amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). The present systems/compositions and methods may be used to modify neurons (e.g., motor neurons) in vitro or in C9orf72 animal models (e.g., mouse models). Targeted DNA methylation editing of C9orf72 can reduce the formation of RNA foci and production of dipeptides to ameliorate the motor neuron degeneration. The present systems can precisely edit the epigenome, including, but not limited to, DNA methylation, histone acetylation, and DNA looping, at one or multiple genomic loci in mammalian cells, both in vitro and in vivo (e.g., in a patient, in an animal model such as mice, etc.). The system may comprise a catalytically dead Cas9 (dCas9) or Cpf1 (dCpf1), fused with one or more effector protein/domain, including, but not limited to, Dnmt3a, Dnmt3b, Tet1, Tet2, p300, and CTCF, that can modify the state of DNA methylation, histone acetylation, DNA looping, etc. The present disclosure provides for a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences. The present disclosure provides for a system comprising: (a) a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain, or a first polynucleotide sequence encoding the fusion protein; and (b) one or more guide sequences that hybridize to one or more target sequences, or a second polynucleotide sequence encoding the one or more guide sequences. In certain embodiments, the effector domain is TET2, Dnmt3b or CTCF. In certain embodiments, the effector domain is CTCF where the polypeptide can modify DNA looping. In certain embodiments, catalytically inactive Cas9 (dCas9) or Cpf1 (dCpf1) is fused with Tet2, Dnmt3b, CTCF, Tet1, Dnmt3a, or p300. In certain embodiments, targeting of the fusion protein to a hypermethylated or unmethylated/hypomethylated promoter, or enhancer, may activate or silence the expression of a gene. Targeted de novo methylation of a CTCF loop anchor site by the fusion protein may block CTCF binding and interfere with DNA looping, which may alter gene expression in the neighboring loop. The target sequence may be located in a non-coding region of a gene. The target sequence may be located in, or near, a promoter, a differentially methylated region (DMR), an enhancer, and/or a CTCF binding site, of a gene. The target sequence may comprise a promoter, a DMR, an enhancer, and/or a CTCF binding site, of a gene. The one or more target sequences (e.g., genomic sequences) may be located within 50 kB of the transcription start site (TSS) of a gene. The target sequence may be located in a coding region of a gene. The target sequence may be located in, or near, a promoter, a differentially methylated region (DMR), an enhancer, and/or a CTCF binding site, of a disease-associated (or disease- related) gene. The target sequence may comprise a promoter, a DMR, an enhancer, and/or a CTCF binding site, of a disease-associated gene (disease- related gene). In certain embodiments, the disease-related gene is C9orf72. The one or more target sequences may be in the C9orf72 gene. The one or more target sequences may be in a non-coding region of the C9orf72 gene. The one or more target sequences may be in a coding region of the C9orf72 gene. The one or more target sequences may be in the GGGGCC (G4C2) repeats in the C9orf72 gene and/or a C9orf72 promoter region. The one or more target sequences may be in CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene. The present system/composition may modify the methylation state of the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region. The present system/composition may modify the methylation state of CGI or CpG-island (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene. The present system/composition may decrease (or increase) the methylation level of the G4C2 repeats in the C9orf72 gene, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration of the present system/composition to a subject and/or cells (or contacting the cells). The present system/composition may decrease (or increase) the methylation level of CGI (e.g., CGI-1 or CGI-2) in the promoter region of the C9orf72 gene, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration of the present system/composition to a subject and/or cells (or contacting the cells). The present system/composition may increase the protein (or transcript) level of C9orf72 by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration of the present system/composition to a subject and/or cells (or contacting the cells). The present system/composition may decrease the level/number of RNA foci in the cells by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration of the present system/composition to a subject and/or cells (or contacting the cells). The present system/composition may decrease the level of poly-dipeptides in the cells by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration of the present system/composition to a subject and/or cells (or contacting the cells). The target sequence may be a genomic sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 target sequences (e.g., genomic sequences) are modified in the cell. The guide sequence may be a CRISPR RNA (crRNA) molecule, a single-guide RNA (sgRNA) molecule, a guide RNA (gRNA), or combinations thereof. The first polynucleotide sequence and the second polynucleotide sequence may be on a single vector, or on different vectors. The second polynucleotide sequence may encode two or more guide sequences that hybridize to two or more target sequences. In certain embodiments, the system contains an all-in-one vector expressing a chimeric protein (or fusion protein), and one crRNA or an array of crRNAs to target the chimeric protein to one or mulitple genomic loci to mediate epigenome editing. The present disclosure provides for a polynucleotide comprising: (a) a first sequence encoding a fusion protein comprising deoxyribonuclease (DNase) dead (or a catalytically dead) nuclease and an effector domain; and (b) a second sequence encoding one, two or more guide sequences that hybridize to one, two or more genomic sequences. The present disclosure provides for a polynucleotide comprising: (a) a first sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead Cas9 (dCas9) or Cpf1 (dCpf1) and an effector domain; and (b) a second sequence encoding one, two or more guide sequences that hybridize to one, two or more genomic sequences. The present disclosure provides a pharmaceutical composition comprising: a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein, a system described herein, or a cell(s) described herein. The present disclosure provides a method of modulating an epigenome of a cell. The method may comprise contacting the cell with the present polynucleotide(s) (nucleic acid(s)), present system, or present composition. In an aspect, the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, the method comprising contacting the cell with the present polynucleotide(s) (nucleic acid(s)), present system, or present composition. The present disclosure provides for a method for modifying an epigenome of a cell. The method may comprise contacting the cell with the present system. In certain embodiments, the methods further comprise introducing the cell into a non- human mammal. The non-human mammal may be a mouse. In another aspect, the disclosure features a method of treating a subject. The method may comprise administering to the subject (or contacting the cell of the subject), an effective amount of the present polynucleotide(s) (nucleic acid(s)), present system, or present composition. The present disclosure provides a method of treating a disease or condition in a subject. The method may comprise administering the present polynucleotide(s) (nucleic acid(s)), present composition, present system, or present cells to the subject. In an embodiment, the subject is an animal or plant. In an embodiment, the subject is a mammal, primate, or human. The present system/method may be used to treat a neurodegenerative disease such as a motor neuron disease (e.g., ALS). The present system/method may be used to treat a neurodegenerative disease such as frontotemporal dementia (FTD). Cell replacement therapy can be used to prevent, correct or treat diseases, where the methods of the present disclosure are applied to isolated patient’s cells (ex vivo), which is then followed by the injection of “corrected” cells back into the patient. For the treatment of a neurological disease, a patient’s iPSC cells may be isolated and differentiated into neurons ex vivo. In certain embodiments, the patient’s iPSC cells or neurons characterized by the different methylation state of DNA (compared to the healthy subjects) in a disease-related gene may be manipulated using methods of the present disclosure in a manner that results in the modification/correction of the DNA methylation state of a disease-related gene. "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as a fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors. The present methods may further comprise differentiating the iPSC cell to a differentiated cell, for example, a neuron. For example, patient fibroblast cells can be collected from the skin biopsy and transformed into iPSC cells. Dimos JT et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321: 1218–1221; Nature Reviews Neurology 4, 582-583 (November 2008). Luo et al., Generation of induced pluripotent stem cells from skin fibroblasts of a patient with olivopontocerebellar atrophy, Tohoku J. Exp. Med. 2012, 226(2): 151-9. The CRISPR-mediated modification can be done at this stage. The corrected cell clone can be screened and selected. The corrected cell clone is then differentiated into, e.g., neurons, and tested for its neuron-specific markers. Well-differentiated neurons can be transplanted autologously back to the donor patient. The cell may be autologous or allogeneic to the subject who is administered the cell. The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the same individual. The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals of the same species are said to be allogeneic to one another. The corrected cells for cell therapy may be administered to a subject. Cells (e.g., neurons) described in the present disclosure may be formulated with a pharmaceutically acceptable carrier. For example, cells can be administered alone or as a component of a pharmaceutical formulation. The cells (e.g., neurons) can be administered in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents. The present disclosure provides for a method for modifying a neurodegenerative disease (such as a motor neuron disease) related gene in a cell. The present disclosure provides for a method for modifying an ALS-related gene in a cell. The present disclosure provides for a method for modifying an FTD-related gene in a cell. The method may comprise contacting the cell with the present system, polynucleotide(s) or composition. The cell may be from a subject having a disease, such as a neurodegenerative disease (for example, a motor neuron disease). The cell may be from a subject having ALS or FTD. The cell may be derived from a cell from a subject having a disease, such as a neurodegenerative disease (for example, a motor neuron disease). The cell may be derived from a cell from a subject having ALS or FTD. The cell may be a stem cell, a neuron, a post-mitotic cell, or a fibroblast. In some aspects, the cell is a human cell or a mouse cell. The cell may be an induced pluripotent stem cell (iPSC), e.g., derived from a fibroblast of a subject. The cell may be an ESC. The method may further comprise culturing the iPSC or ESC to differentiate into, e.g., a neuron. The method may further comprise administering the differentiated cell (e.g., a neuron) to a subject. The cell may be autologous or allogeneic to the subject. The present disclosure provides for a method for treating a neurodegenerative disease (including a motor neuron disease) in a patient. The method may comprise administering the present system to the patient. The present polypeptide(s)/system may be used in a method for modifying an epigenome of a cell or a genomic sequence in a cell. The method comprises contacting the cell with the present system/polynucleotide(s). The genomic sequence may be any suitable genomic sequence. The present systems/methods may allow precise gene activation or silencing. The present systems/methods may enable multiplex editing of more than one genomic locus. The present systems/methods can allow epigenome editing at multiple sites using a single vector. U.S. Patent Publication No. 20190359959 is incorporated by reference herein in its entirety. The present disclosure provides for a method for modifying an X-linked disease-related gene or an imprinting-related disease-related gene in a cell. In certain embodiments, the present systems/methods can be used to treat a disorder/disease. For example, the systems/methods can be applied to reactivate the wild type allele of a gene associated with an X-linked disease, or a gene associated with an imprinting-related disease, via epigenetic editing. In certain aspects, one or more of the target sequences (e.g., genomic sequences) are associated with a disease or condition. The present system may target a target sequence that is associated with a disease-related gene, such as a gene associated with a neurodegenerative disease, a gene associated with a motor neuron disease, a gene associated with an X-linked disease, or a gene associated with an imprinting- related disease. In certain aspects, the method may further comprise administering to the subject an agent that inhibits or enhances DNA methylation. The agent may be a small molecule. For example, the agent is 5-azacytidine or 5-azadeoxycytidine. In certain aspects, the method may further comprise contacting the cell with an agent that inhibits or enhances DNA methylation. The agent may be a small molecule. For example, the agent is 5-azacytidine or 5-azadeoxycytidine. The present disclosure provides for a method of modifying a disease-related gene. The method may comprise introducing the present system/polynucleotide(s) into a cell. The present disclosure provides for gene editing methods that can modify the disease- related gene, which in turn can be used for in vivo gene therapy for patients afflicted with the disease. Furthermore, methods of the present disclosure may be applied to specific gene- humanized mouse model as well as patient-derived cells, allowing for determining the efficiency and efficacy of designed sgRNA and site-specific recombination frequency in human cells, which can be then used as a guide in a clinical setting. The present disclosure provides a cell comprising: a system described herein, a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein; or a composition described herein. The cell may be a vertebrate, mammalian (e.g., human), rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, or primate, cell. The cell may be a plant cell. In an embodiment, the cell is a human cell. The cell may be somatic cells, stem cells, mitotic or post-mitotic cells, neurons, fibroblasts, or zygotes. A cell, zygote, embryo, or post-natal mammal can be of vertebrate (e.g., mammalian) origin. In some aspects, the vertebrates are mammals or avians. Particular examples include primate (e.g., human), rodent (e.g., mouse, rat), canine, feline, bovine, equine, caprine, porcine, or avian (e.g., chickens, ducks, geese, turkeys) cells, zygotes, embryos, or post-natal mammals. In some embodiments, the cell, zygote, embryo, or post-natal mammal is isolated (e.g., an isolated cell; an isolated zygote; an isolated embryo). In some embodiments, a mouse cell, mouse zygote, mouse embryo, or mouse post-natal mammal is used. In some embodiments, a rat cell, rat zygote, rat embryo, or rat post-natal mammal is used. In some embodiments, a human cell, human zygote or human embryo is used. The cell may be a somatic cell, germ cell, or prenatal cell. The cell may be a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell. The present disclosure provides a kit comprising: a polypeptide(s) described herein; a nucleic acid(s) described herein; a vector(s) described herein; a system described herein, or a composition described herein. The kit may comprise an instruction for using the system, the polypeptide(s), the nucleic acid(s), the vector(s), or the composition, in a method described herein. Cas enzymes The Cas enzyme of the CRISPR/Cas system may be Cas9, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, 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, Cpf1, homologs thereof, orthologs thereof, or modified versions thereof. In one embodiment, the Cas enzyme is Cas9. Cas9 is a CRISPR associated endonuclease. Non-limiting exemplary Cas9s are provided herein, e.g. the Cas9 provided for in UniProtKB G3ECR1 (CAS9_STRTR) or the Staphylococcus aureus Cas9, as well as the nuclease dead Cas9, orthologs and biological equivalents each thereof. Orthologs include but are not limited to Streptococcus pyogenes Cas9 (“spCas9”); Cas 9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida; and Cpf1 (which performs cutting functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112. A nuclease-defective or nuclease-deficient Cas protein (e.g., dCas9) with one or more mutations on its nuclease domains retains DNA binding activity when complexed with a guide sequence (e.g., gRNA). dCas protein can tether and localize effector domains or protein tags by means of protein fusions to sites matched by a guide sequence (e.g., gRNA), thus constituting an RNA-guided DNA binding enzyme. In some embodiments, the nucleotide sequence encoding the Cas (e.g., Cas9) nuclease is modified to alter the activity of the protein. In some embodiments, the Cas (e.g., Cas9) nuclease is a catalytically inactive Cas (e.g., Cas9) (or a catalytically deactivated/defective Cas9 or dCas9). In one embodiment, dCas (e.g., dCas9) is a Cas protein (e.g., Cas9) that lacks endonuclease activity due to point mutations at one or both endonuclease catalytic sites (RuvC and HNH) of wild type Cas (e.g., Cas9). For example, dCas9 contains mutations of catalytically active residues (D10 and H840) and does not have nuclease activity. In some cases, dCas has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA. As a non-limiting example, in some cases, dCas9 harbors both D10A and H840A mutations of the amino acid sequence of S. pyogenes Cas9. In some embodiments when a dCas9 has reduced or defective catalytic activity (e.g., when a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or a A987 mutation (or combinations thereof), e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A(or combinations thereof)), the Cas protein can still bind to target DNA in a site-specific manner, because it is still guided to a target polynucleotide sequence by a DNA-targeting sequence of the subject polynucleotide (e.g., gRNA), as long as it retains the ability to interact with the Cas-binding sequence of the subject polynucleotide (e.g., gRNA). The nuclease may be a deoxyribonuclease (DNase) dead (or a catalytically dead) Cas9 (e.g., spCas9). The catalytically dead Cas9 (dCas9) may contain one or more of the following mutations: D10A and H840A. In certain embodiments, the DNase catalytically dead Cas9 (dCas9) or Cpf1 (dCpf1) has RNase activity. Cpf1 may be from Flavobacterium brachiophilum, Parcubacteria bacterium, Peregrinibacteria bacterium, Acidaminococcus sp., Porphyromonas macacae, Lachnospiraceae bacterium, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida, Candidatus methanoplasma termitum, or Eubacterium eligens. In one embodiment, dCpf1 is catalytically dead LbCpf1 (from Lachnospiraceae bacterium). In another embodiment, dCpf1 is catalytically dead AsCpf1 (from Acidaminococcus sp.). In yet another embodiment, dCpf1 is catalytically dead FbCpf1 (from Flavobacterium brachiophilum). AsCpf1 may have the UniProt number UniProtKB-U2UMQ6 (CS12A_ACISB), and comprise the corresponding amino acid sequence. LbCpf1 may have the UniProt number UniProtKB-A0A182DWE3 (A0A182DWE3_9FIRM), and comprise the corresponding amino acid sequence. The nuclease may be a deoxyribonuclease (DNase) dead (or a catalytically dead) Cpf1 (dCpf1). The dCpf1 may comprise one or more of the following mutations: D908A, E993A, R1226A and D1263A. The dCpf1 may be Cpf1 comprising the following mutation: D833A. There may be a number of different isoforms for each of these proteins/polypeptides discussed in this disclosure, provided herein are the general accession numbers, NCBI Reference Sequence (RefSeq) accession numbers, GenBank accession numbers, and/or UniProt numbers to provide relevant sequences. The proteins/polypeptides may also comprise other sequences. In all cases where an accession number (e.g., a UniProt number) is used, the accession number refers to one embodiment of the protein or gene which may be used with the systems/methods of the present disclosure. Effector domains The effector domain may have an activity to modify the epigenome of a cell. The effector domain may be a molecule (e.g., protein or a polypeptide) that modulates the expression and/or activation of a genomic sequence (e.g., gene). In some aspects, the effector domain modifies one or both alleles of a gene. The effector domain can be introduced as a nucleic acid sequence and/or as a protein. In some aspects, the effector domain can be a constitutive or an inducible effector domain. In some aspects, a Cas (e.g., dCas9, dCpf1, etc.) nucleic acid sequence or variant thereof and an effector domain nucleic acid sequence are introduced into the cell as a chimeric sequence. In some aspects, the effector domain is fused to a molecule that associates with (e.g., binds to) Cas protein (e.g., the effector molecule is fused to an antibody or antigen binding fragment thereof that binds to Cas protein). In some aspects, a Cas (e.g., dCas9, dCpf1, etc.) protein or variant thereof and an effector domain are fused or tethered creating a chimeric protein and are introduced into the cell as the chimeric protein. In some aspects, the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain bind due to a protein- protein interaction. In some aspects, the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain are covalently linked. In some aspects, the effector domain associates non-covalently with the Cas (e.g., dCas9, dCpf1, etc.) protein. In some aspects, a Cas (e.g., dCas9, dCpf1, etc.) nucleic acid sequence and an effector domain nucleic acid sequence are introduced as separate sequences and/or proteins. In some aspects, the Cas (e.g., dCas9, dCpf1, etc.) protein and effector domain are not fused or tethered. As shown herein, fusions of a catalytically inactive Cas protein (e.g., dCas9, dCpf1, etc.) tethered with all or a portion of (e.g., biologically active portion of) an (one or more) effector domain create chimeric proteins that can be guided to specific DNA sites by one or more guide sequences, to modulate activity and/or expression of one or more genomic sequences (e.g., exert certain effects on transcription or chromatin organization, or bring specific kind of molecules into specific DNA loci, or act as sensor of local histone or DNA state). In specific aspects, fusions of dCas9 (or dCpf1) tethered with all or a portion of an effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences to modulate or modify methylation or demethylation of one or more genomic sequences. As used herein, a "biologically active portion of an effector domain" is a portion that maintains the function (e.g., completely, partially, minimally) of an effector domain (e.g., a "minimal" or "core" domain). The effector domain may be an enzyme that modifies methylation state of DNA. The effector domain may have methylation activity or demethylation activity (e.g., DNA methylation or DNA demethylation activity). For example, the effector domain may be a DNA methyltransferase (DNMT, such as Dnmt3b and Dmnt3a) or a Ten-Eleven-Translocation (TET) methylcytosine dioxygenase protein (such as Tet2 or Tet1). The effector domain may be ACIDA, MBD4, Apobec1, Apobec2, Apobec3, Tdg, Gadd45a, Gadd45b, or ROS1. The effector domain may be Dnmt1, Dnmt3a, Dnmt3b, CpG Methyltransferase M.SssI, or M.EcoHK3 II. The effector domain may be an enzyme that modifies a histone subunit, such as a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase (e.g., LSD1). In one embodiment, the HAT is p300. The effector domain may be CTCF, including wild type CTCF or a DNA binding mutant CTCF. In certain embodiments, the DNA binding mutant CTCF comprises one or more of the following mutations: K365A, R368A, R396A, and Q418A. The effector domain may be a transcriptional activation domain, such as a transcriptional activation domain derived from VP64, VPR or NF-κB p65. The effector domain may be a transcriptional silencer (heterochromatin protein 1 (HP1), or Methyl CpG binding Protein 2 (MeCP2)) or transcriptional repression domain (e.g., a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID)). Examples of effector domains also include a transcription(al) activating domain, a coactivator domain, a transcription factor, a transcriptional pause release factor domain, a negative regulator of transcriptional elongation domain, a transcriptional repressor domain, a chromatin organizer domain, a remodeler domain, a histone modifier domain, a DNA modification domain, and an RNA binding domain. Other examples of effector domains include histone marks readers/interactors and DNA modification readers/interactors. In one aspect of the invention, fusion of the dCas9 (or dCpf1) to an effector domain can be to that of a single copy or multiple/tandem copies of full-length or partial-length effector domains. Other fusions can be with split (functionally complementary) versions of the effector domains. Other examples of effector domains are described in PCT Publication No. WO2014172470 and U.S. Publication No. US20160186208, which are incorporated herein by reference in their entirety. In some aspects, the Cas (e.g., dCas9, dCpf1, etc.) protein can be fused to the N-terminus or C-terminus of the effector domain. In one aspect, fusion of dCas9 (or dCpf1) with all or a portion of one or more effector domains comprise one or more linkers. In one aspect, a linker comprises one or more amino acids. In some aspects, a linker comprises two or more amino acids. In one aspect, a linker comprises the amino acid sequence GS. In some aspects, fusion of Cas (e.g., dCas9, dCpf1, etc.) with two or more effector domains comprises one or more interspersed linkers (e.g., GS linkers) between the domains. In some aspects, one or more nuclear localization sequences (NLS) may be located between the catalytically inactive nuclease (e.g., dCas9, dCpf1, etc.) and the effector domain. Target sequences In some embodiments, a target sequence is located within or near an essential gene or a non-essential gene. In an embodiment, the target sequence may be derived from a gene (e.g., a disease-related gene) described herein. In some aspects, one copy of one or more genomic sequences in the cell is modified. In some aspects, both copies of one or more of the genomic sequences in the cell are modified. In some aspects, the one or more genomic sequences that are modified are endogenous to the cell. In particular aspects, at least two of the genomic sequences are endogenous genomic sequences. In some aspects, at least two of the genomic sequences are exogenous genomic sequences. In some aspects where there are at least two genomic sequences, at least one of the genomic sequences is an endogenous genomic sequence and at least one of the genomic sequences is an exogenous genomic sequence. In some aspects, at least two of the genomic sequences are endogenous genes. In some aspects, at least two of the genomic sequences are exogenous genes. In some aspects where there are at least two genomic sequences, at least one of the genomic sequences is an endogenous gene and at least one of the genomic sequences is an exogenous gene. In some aspects, at least two of the genomic sequences are at least 1 kB apart. In some aspects, at least two of the genomic sequences are on different chromosomes. The target sequence may comprise a differentially methylated region (DMR). A differentially methylated region may be differentially methylated between cells of different cell types (e.g., muscle cells vs neuron, or skin cells vs hepatocytes). A differentially methylated region may be differentially methylated between diseased vs non-diseased cells (e.g., cancer vs non- cancer cells). A differentially methylated region may be differentially methylated between differentiation states (e.g., progenitor cells vs terminally differentiated cells). The effect on expression of one or more genes (e.g., within up to about 0.5, 1, 2, 5, 10, 20, 50, 100, 500 kb or within about 1, 2, 5, or 10 MB from the modification) may be assessed. In some aspects, the differentially methylated region may be hypermethylated, unmethylated or hypomethylated. Also disclosed are methods of modulating the expression of one or more genes of interest in a cell, wherein a differentially methylated region is located within 50 kB of the transcription start site of the gene. The method may comprise contacting the cell with the present system, where the guide sequence targets the differentially methylated region. In some aspects, the differentially methylated region is hypermethylated in the cell and the effector domain (e.g., Tet2 or Tet1) has demethylation activity. In other aspects, the differentially methylated region is unmethylated or hypomethylated in the cell and the effector domain (e.g., Dnmt3a, Dnmt3b) has methylation activity. In some aspects, the present system/method may demethylate a genomic sequence that is aberrantly hypermethylated, or may methylate a genomic sequence that is aberrantly unmethylated or hypomethylated. In some aspects, an aberrantly hypermethylated sequence, or aberrantly unmethylated or hypomethylated sequence, may occur in a disease or disorder. In other aspects, it is of interest to methylate a CTCF site (e.g., a CTCF binding site) that is aberrantly unmethylated or hypomethylated, or remove methylation of a CTCF site that is aberrantly methylated. Modifying the methylation or demethylation of the CTCF site may treat or prevent a disease or disorder that exhibits an aberrantly unmethylated or hypomethylated sequence or region, or an aberrantly hypermethylated sequence or region. For example, a CTCF loop may be opened by methylating a CTCF binding site and thereby bring a gene that is outside the loop under control of an enhancer inside the loop if one wanted to increase expression of that gene (e.g., if expression of the gene is aberrantly low and/or if increased expression is desired for therapeutic or other purposes). In some aspects, the present system/method may modify a promoter sequence. Targeting of the present system to methylated, hypermethylated, unmethylated or hypomethylated promoter sequences may cause activation or silencing of expression of a gene. In some aspects, the present system/method may modify an enhancer sequence. Targeting of the present system to methylated, hypermethylated, unmethylated or hypomethylated enhancer sequences may cause activation or silencing of expression of a gene. In some aspects, the present system/method may modify a CTCF binding site. Targeting of the present system to CTCF binding sites may affect CTCF binding and interfere with, or increase, DNA looping, which may alter gene expression (e.g., in the neighboring loop). In one aspect, multiple genomic sequences are modulated (e.g., multiplexed activation). All or a portion of the region targeted by the one or more guide sequences may be a differentially methylated region. In some aspects, the differentially methylated region is exactly or within about 25 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1500 bases, 2000 bases, 5000 bases, 10000 bases, 20000 bases, 50000 bases or more upstream to the one or more genes (e.g., endogenous genes; exogenous genes) or a (one or more) transcription start site (TSS). In some aspects, the differentially methylated region is exactly or within about 25 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1500 bases, 2000 bases, 5000 bases, 10000 bases, 20000 bases, 50000 bases, or more downstream to the one or more genes (e.g., endogenous genes; exogenous genes) or a TSS. The regulatory region targeted by one or more guide sequences may be entirely or partially found at or about the 5’ end of the gene (e.g., endogenous or exogenous) or a TSS. The 5’ end of a gene can include un- transcribed (flanking) regions (e.g., all or a portion of a promoter) and a portion of the transcribed region. "Modulate" or "modify" means to cause or facilitate a qualitative and/or quantitative change, alteration, or modification in a level (expression level), an activity, a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon. The present system/method may result in an increase of the expression level or activity of at least one (wildtype) gene or protein, or a decrease of the expression level or activity of at least one (mutant) gene or protein, by at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99%, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames following administration to a subject and/or cells (or contacting the cells). The expression level and/or activity of the (wildtype) gene or protein may increase, or the expression level and/or activity of the (mutant) gene or protein may decrease, by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.5 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 15 fold, at least or about 20 fold, at least or about 50 fold, at least or about 100 fold, at least or about 120 fold, from about 2 fold to about 500 fold, from about 1.1 fold to about 10 fold, from about 1.1 fold to about 5 fold, from about 1.5 fold to about 5 fold, from about 2 fold to about 5 fold, from about 3 fold to about 4 fold, from about 5 fold to about 10 fold, from about 5 fold to about 200 fold, from about 10 fold to about 150 fold, from about 10 fold to about 20 fold, from about 20 fold to about 150 fold, from about 20 fold to about 50 fold, from about 30 fold to about 150 fold, from about 50 fold to about 100 fold, from about 70 fold to about 150 fold, from about 100 fold to about 150 fold, from about 10 fold to about 100 fold, or from about 100 fold to about 200 fold, in about 2 hours, in about 5 hours, in about 10 hours, in about 24 hours, in about 1 day, in about 2 days, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 1 week, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks, in about 1 month, in about 2 months, in about 3 months, in about 4 months, in about 5 months, in about 6 months, from about 1 week to about 2 weeks, or within different time-frames, following administration to a subject and/or cells (or contacting the cells). Guide sequences The gRNA (or crRNA, or sgRNA) may contain a targeting segment that can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target sequence ("target region" or "target DNA"). In certain embodiments, the gRNA (or crRNA, or sgRNA) sequence (or the targeting segment of the gRNA (or crRNA, or sgRNA)) has 100% complementarity to the target sequence. The targeting segment of the gRNA (or crRNA, or sgRNA) may have full complementarity with the target sequence. The targeting segment of the gRNA (or crRNA, or sgRNA) may have partial complementarity with the target sequence. In certain embodiments, the targeting segment of the gRNA (or crRNA, or sgRNA) has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the target sequence (mismatches). In certain embodiments, the gRNA (or crRNA, or sgRNA) is about 10 nucleotides to about 150 nucleotides in length. In certain embodiments, the targeting segment of the gRNA (or crRNA, or sgRNA) is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides in length. In certain embodiment, the targeting segment of the gRNA (or crRNA, or sgRNA) is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In certain embodiments, the targeting segment of the gRNA (or crRNA, or sgRNA) is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length. In one embodiment, the degree of complementarity, together with other properties of the gRNA (or crRNA, or sgRNA), is sufficient to allow targeting of a Cas molecule to the target nucleic acid. The term “gRNA” or “guide RNA” as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique. Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature biotechnology 2014; 32(12):1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38, and Graham, D., et al. Genome Biol. 2015; 16: 260. gRNA may comprise, or alternatively consist essentially of, or yet further consist of, a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA). In some aspects, a gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83). As used herein, a biological equivalent of a gRNA includes but is not limited to polynucleotides or targeting molecules that can guide a Cas or equivalent thereof to a specific nucleotide sequence such as a specific region of a cell’s genome. The guide sequence (e.g., crRNA, sgRNA, gRNA, etc.) used in the present system/method can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 5960, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 9192, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer). In one embodiment, the guide sequence (e.g., crRNA, sgRNA, gRNA, etc.) can be between about 15 and about 30 nucleotides in length (e.g., about 15-29, 15-26, 15-25; 16-30, 16-29, 16-26, 16-25; or about 18-30, 18-29, 18-26, or 18-25 nucleotides in length). As described herein, the one or more guide sequences also comprise a (one or more) binding site for a (one or more) catalytically inactive site-specific nuclease. The catalytically inactive site-specific nuclease may be a catalytically inactive CRISPR associated (Cas) protein, such as dCas9, and dCpf1. In a particular aspect, upon hybridization of the one or more guide sequences to the one or more target sequences, the catalytically inactive site-specific nuclease binds to the one or more guide sequences. In certain embodiments, the guide sequence is an RNA sequence. In one aspect, a single RNA sequence can be complementary to one or more (e.g., all) of the genomic sequences that are being modulated or modified. In one aspect, a single RNA is complementary to a single target genomic sequence. In a particular aspect in which two or more target genomic sequences are to be modulated or modified, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) RNA sequences are used wherein each RNA sequence is complementary to (specific for) one target genomic sequence. In some aspects, two or more, three or more, four or more, five or more, or six or more RNA sequences are complementary to (specific for) different parts of the same target sequence. In one aspect, two or more RNA sequences bind to different sequences of the same region of DNA. In some aspects, a single RNA sequence is complementary to at least two target or more (e.g., all) of the genomic sequences. It will also be apparent to those of skill in the art that the portion of the RNA sequence that is complementary to one or more of the genomic sequences and the portion of the RNA sequence that binds to the catalytically inactive site-specific nuclease can be introduced as a single sequence or as 2 (or more) separate sequences into a cell, zygote, embryo or nonhuman animal. In some embodiments, the sequence that binds to the catalytically inactive site-specific nuclease comprises a stem-loop. In certain embodiments, the system contains one or more guide sequences (or a polynucleotide sequence encoding one or more guide sequences) that are complementary to all or a portion of a (one or more) regulatory region, an open reading frame (ORF; a splicing factor), an intronic sequence, a chromosomal region (e.g., telomere, centromere) of the one or more genomic sequences in a cell. In some aspects, the regulatory region targeted by one or more genomic sequences is a promoter, enhancer, and/or operator region. In some aspects, all or a portion of the regulatory region is targeted by the one or more guide sequences. gRNAs can be generated to target a specific gene, optionally a gene associated with a disease, disorder, or condition. Thus, in combination with Cas, the guide RNAs facilitate the target specificity of the CRISPR/Cas system. Further aspects such as promoter choice, as discussed herein, may provide additional mechanisms of achieving target specificity – e.g., selecting a promoter for the guide RNA encoding polynucleotide that facilitates expression in a particular organ or tissue. Accordingly, the selection of suitable gRNAs for the particular disease, disorder, or condition is contemplated herein. Conditions to be treated The present disclosure provides for a method for treating a neurodegenerative disease (such as a motor neuron disease) in a subject. Motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome. The method may comprise administering to the subject a therapeutically effective amount of the present system, polynucleotide(s) or composition. The terms "disease", "disorder" or "condition" are used interchangeably and may refer to any alteration from a state of health and/or normal functioning of an organism, e.g., an abnormality of the body or mind that causes pain, discomfort, dysfunction, distress, degeneration, or death to the individual afflicted. In some embodiments, a disease is a psychiatric, neurological, neurodevelopmental disease, neurodegenerative disease, cardiovascular disease, autoimmune disease, cancer, metabolic disease, or respiratory disease. In some embodiments a disease is a psychiatric, neurological, or neurodevelopmental disease, e.g., schizophrenia, depression, bipolar disorder, epilepsy, autism, addiction. Neurodegenerative diseases include, e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). In some embodiments, a disease exhibits hypermethylation (e.g., aberrant hypermethylation) or unmethylation/hypomethylation (e.g., aberrant unmethylation or hypomethylation) in a genomic sequence. The methods described herein may be used to treat or prevent diseases or disorders exhibiting aberrant methylation (e.g., hypermethylation or unmethylation/hypomethylation). As used herein, the terms “neurodegenerative disease”, “neurodegenerative disorder”, and “neurodegenerative condition” generally refer to any disease, disorder, and/or condition that affects the neurons (sometimes referred to as “nerve cells”), such as neurons of a brain and/or neurons of a nervous system which is associated with the degeneration or loss of neural cells. Neurodegenerative diseases may result in progressive degeneration and/or death of nerve cells. In general, neurodegeneration is the progressive loss of structure and/or function of neurons, including the death of neurons. Neurodegenerative diseases may cause problems with movement (e.g., ataxias), or mental or cognitive functioning (e.g., dementias). Frequently neurodegeneration is associated with neuroinflammation. Therefore, it is to be understood that neurodegenerative diseases or disorders encompass neural diseases which are characterized by neuroinflammation. Sometimes in such diseases activated microglia may produce inflammatory cytokines that contribute to widespread inflammation and may lead to and/or result in a neurodegenerative condition and/or disease. Some neurodegenerative diseases and/or conditions are associated with microglia cell over-activation, increased numbers of microglia cells, production of inflammatory proteins and/or inflammatory activities, and/or neuronal death. In some embodiments, the present method/system (or composition) may decrease or prevent at least one symptom associated with a neurodegenerative disease. The present system/composition and method may result in a decrease in neurodegeneration, degeneration of neurons (e.g., degeneration of motor neurons), the loss of neurons, neuronal cell death, morphological abnormalities of the neuromuscular junctions (NMJs), etc. of the subject, where neurodegeneration, degeneration of neurons, the loss of neurons, neuronal cell death, morphological abnormalities of the neuromuscular junctions (NMJs), etc. of the subject affected by the present system/composition and method is no greater than 90%, no greater than 85%, no greater than 80%, no greater than 75%, no greater than 70%, no greater than 65%, no greater than 60%, no greater than 55%, no greater than 50%, no greater than 45%, no greater than 40%, no greater than 35%, no greater than 30%, no greater than 25%, no greater than 20%, no greater than 15%, no greater than 10%, about 10% to about 90%, about 15% to about 80%, about 20% to about 70%, about 25% to about 60%, about 30% to about 50%, about 30% to about 40%, about 25% to about 40%, about 20% to about 30%, about 25% to about 35%, about 10% to about 30%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 20% to about 50%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, of the neurodegeneration, the degeneration of neurons, the loss of neurons, neuronal cell death, morphological abnormalities of the neuromuscular junctions (NMJs), etc. of the subject in the absence of the present system/composition and method. The present method and system/composition may ameliorate the symptoms of a neurodegenerative disease or disorder in a subject. The present method and system/composition may result in at least partial correction of neuropathology, and/or alleviation and/or prevention and/or stabilization and/or slowing of disease progression, and/or progression of the symptoms of a neurodegenerative disease or disorder. The present method and composition may prevent neuron death, and/or delay the onset of paralysis and death. The present system/composition and method may result in an increase in motor neuron number, neuromuscular junction (NMJ) electrophysiology (e.g., miniature end-plate potentials (mEPPs), end-plate potentials (EPPs), Quantal content), neurotransmission at the NMJ, muscle strength, etc. of the subject, where the motor neuron number, neuromuscular junction (NMJ) electrophysiology (e.g., miniature end-plate potentials (mEPPs), end-plate potentials (EPPs), Quantal content), neurotransmission at the NMJ, muscle strength, etc. of the subject affected by the present composition and method is at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 1.1-fold, at least or about 1.2-fold, at least or about 1.3-fold, at least or about 1.4-fold, at least or about 1.5-fold, at least or about 1.6-fold, at least or about 1.8-fold, at least or about 15-fold, at least or about 20-fold, at least or about 50-fold, at least or about 100-fold, at least or about 120-fold, from about 2-fold to about 500-fold, from about 1.1-fold to about 10-fold, from about 1.1-fold to about 5-fold, from about 1.5-fold to about 5-fold, from about 2-fold to about 5-fold, from about 3-fold to about 4- fold, from about 5-fold to about 10-fold, from about 5-fold to about 200-fold, from about 10-fold to about 150-fold, from about 10-fold to about 20-fold, from about 20-fold to about 150-fold, from about 20-fold to about 50-fold, from about 30-fold to about 150-fold, from about 50-fold to about 100-fold, from about 70-fold to about-150 fold, from about 100-fold to about 150-fold, from about 10-fold to about 100-fold, from about 100-fold to about 200-fold, of the motor neuron number, neuromuscular junction (NMJ) electrophysiology (e.g., miniature end-plate potentials (mEPPs), end-plate potentials (EPPs), Quantal content), neurotransmission at the NMJ, muscle strength, etc. of the subject in the absence of the present composition and method. The methods of the present disclosure may be used to treat patients at a different stage of the disease (e.g., early, middle or late). The present methods may be used to treat a patient once or multiple times. Thus, the length of treatment may vary and may include multiple treatments. Delivering the present systems The nuclease (e.g., dCas9, dCpf1, etc.) can be introduced into the cell in the form of DNA, mRNA or protein. The sequence-specific nuclease can be introduced into the cell in the form of a protein or in the form of a nucleic acid encoding the sequence-specific nuclease, such as an mRNA or a cDNA. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics. As an example, CRISPR/Cas may be encoded by a viral vector. The polynucleotide/vector may be a recombinant lentiviral vector, or an adeno-associated viral (AAV) vector, such as an AAV2 vector, or an AAV8 vector. A variety of viral constructs may be used to deliver the present system to the targeted cells and/or a subject. Non-limiting examples of such recombinant viruses include recombinant lentiviruses, recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids, and phages. Options for gene delivery viral constructs are well known (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71). AAV viral vectors may be selected from among any AAV serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or other known and unknown AAV serotypes. In certain embodiments, AAV2 and/or AAV8 are used. The term AAV covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise. Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome of a second serotype. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used as an alternative to viral vectors. Further examples of alternative delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1;459(1-2):70-83). Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue- specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Moreover, inducible and tissue specific expression of an RNA, transmembrane proteins, or other proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter/regulatory sequence. Examples of tissue-specific or inducible promoter/regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer/promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto. Vectors according to the present disclosure can be transformed, transfected or otherwise introduced into a wide variety of host cells. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome. The present system may be delivered by any suitable means. In certain embodiments, the system is delivered in vivo. In other embodiments, the system is delivered to isolated/cultured cells (e.g., iPSC cells) in vitro to provide modified cells useful for in vivo delivery to a subject/patient. In one embodiment, the disclosure provides for introducing the present system or composition into a eukaryotic cell. The cell may be a stem cell. Examples of stem cells include pluripotent, totipotent, multipotent and unipotent stem cells. Examples of pluripotent stem cells include embryonic stem cells, embryonic germ cells, fetal stem cells, adult stem cells, embryonic carcinoma cells and induced pluripotent stem cells (iPSCs). The cell may be a somatic cell. Somatic cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line capable of prolonged proliferation in culture (e.g., for longer than 3 months) or indefinite proliferation (immortalized cells). Adult somatic cells may be obtained from individuals, e.g., human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art. Somatic cells of use in aspects of the invention include mammalian cells, such as, for example, human cells, non-human primate cells, or rodent (e.g., mouse, rat) cells. They may be obtained by well-known methods from various organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, breast, reproductive organs, muscle, blood, bladder, kidney, urethra and other urinary organs, etc., generally from any organ or tissue containing live somatic cells. Mammalian somatic cells useful in various embodiments include, for example, fibroblasts, Sertoli cells, granulosa cells, neurons, pancreatic cells, epidermal cells, epithelial cells, endothelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc. The present system or composition may be introduced into a cell, a zygote, an embryo, a human subject, or a non-human mammal. In an embodiment, the cell is a cancer cell or other cell characterized by a disease or disorder. In an embodiment, the target sequence is derived from the nucleic acid of a human cell. In an embodiment, the target sequence is derived from the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In an embodiment, the cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In an embodiment, the cell is a cell characterized by an unwanted genomic component (e.g., a viral genomic component), such as a cell infected with viruses, a cell infected with bacteria etc. Subjects, which may be treated according to the present disclosure, include all animals which may benefit from the present invention. Such subjects include mammals, preferably humans (infants, children, adolescents and/or adults), but can also be an animal such as dogs and cats, farm animals such as cows, pigs, sheep, horses, goats and the like, and laboratory animals (e.g., rats, mice, guinea pigs, and the like). The present disclosure provides a pharmaceutical composition comprising the present system. The pharmaceutical composition may contain a pharmaceutically and/or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is balanced salt solution. In one embodiment, the carrier includes Tween. If the virus is to be stored long-term, it may be frozen in the presence of glycerol or Tween-20. The present system, cells or compositions may be administered by, direct delivery to a desired organ or tissue, injection, oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Additionally, routes of administration may be combined, if desired. Administration may be through any suitable routes, including but not limited to, intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, and inhalation. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. It is noted that dosage may be impacted by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10% or 20%. As used herein, “treating” or “treatment” of a disease or a condition in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, the term “treatment” excludes prevention. The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Example 1 We hypothesize that the methylation of the C9orf72 locus represents a critical factor in determining the onset and progression of signs and symptoms of ALS in C9orf72 mutant carriers. We applied the dCas9-Tet1 tool to edit the DNA methylation of the G4C2 repeats and C9orf72 promoter, and characterize the edited C9-ALS iPSC and derived motor neurons. We observed RNA foci was abolished in G4C2 repeat deletion line and the neuronal activity was elevated in motor neurons derived from this line. We also established two lines of C9 BAC mouse lines carrying human BAC (bacterial artificial chromosome) C9orf72 with G4C2 repeat expansion mutant. We detected the disease hall marker for C9ALS, poly glycine–arginine dipeptide (GR) in the brain section of C9 mice. Specifically, we have generated two isogenic pairs of C9orf72 ALS/FTD iPSCs. Each pair includes a patient iPSC line with hundreds of G4C2 repeats and a control line in which the expanded G4C2 repeats are deleted. We also generated lentiviral vectors to deliver DNA methylation editors including dCas9-Dnmt3a, dCas9-Tet1, target sgRNAs, and scrambled sgRNA in vitro and in vivo. We have performed DNA methylation editing of the G4C2 repeats and CGIs in C9orf72 ALS/FTD iPSCs and isogenic controls. The editing efficiency has been examined by Pyro-seq to evaluate the targeted methylation. We have been examining the potential off-target effect at DNA methylation and transcription levels. In addition, we have established isoform- specific qPCR assay allowing for the measurement of three C9ofr72 isoforms expression, RNA- FISH method to examine the RNA foci containing G4C2 repeats, and immunofluorescence and blotting to examine the dipeptide repeat protein. Importantly, we observed that targeted DNA methylation of the G4C2 repeats reduced the formation of RNA foci, one of the molecular hallmarks for C9-ALS/FTD pathology. Results We carried out studies to dissect the function of CGI and G4C2 methylation during the degeneration of ALS/FTD neurons. First, we have generated two isogenic pairs of C9orf72 ALS/FTD iPSCs using CRISPR genome editing technology, and each pair includes a patient iPSC line and a control line in which the expanded G4C2 repeats are deleted as validated by primed- repeat PCR as illustrated in Figures 1A and 1B. In addition, we obtained another two isogenic pairs of C9orf72-ALS/FTD iPSCs lines (Figure 1C). In total, we have four isogenic pairs of C9orf72-ALS/FTD iPSCs lines for our studies. Second, characterization of these cells showed that the C9orf72 protein is reduced in patient cells compared to wild-type control (Figure 1D and 1E) as well as the C9orf72 transcripts (Figure 1F). To examine the methylation status of C9orf72 locus, we performed Pyro-seq. The result in Figure 1G showed hypermethylation (~50%) of the CGI-1 in patient iPSCs and hypomethylation (< 3%) in isogenic control with G4C2 repeats deletion, suggesting the association of G4C2 repeat expansion and hypermethylation of CGI-1 at the pathological allele of C9orf72 gene. We collected 9 antibodies against dipeptide repeat proteins (DPRs) including anti-Poly-GR, anti-Poly-GP, anti-Poly-GA, anti-Poly-PA, anti-Poly-PR to examine these cells. We were able to validate these antibodies in HEK293T cells with overexpressed DPR constructs, but we did not detect any of these DPRs in C9orf72 patients iPSCs (Figure 1H). Last, we performed RNA FISH to examine RNA foci containing G4C2 repeat. As shown in Figure 1I, we detected 6% of patient iPSCs with more than two RNA foci, whereas the isogenic control without G4C2 repeats only show 1% of foci-positive cells. These characterizations of C9orf72-ALS/FTD iPSCs and isogenic controls lay down a solid base to perform DNA methylation editing and evaluate the functional consequence of DNA methylation to neurodegeneration. Next, we performed DNA methylation editing of the G4C2 repeats in C9orf72 ALS/FTD iPSCs with isogenic controls. As shown in Figure 2A, we used lentiviral vectors to express dCas9- Dnmt3a-P2A-GFP and sgRNA-mCherry in C9orf72-ALS/FTD iPSC line #52 with about 800 G4C2 repeats and isolated the infection-positive (GFP+;mCherry+) population by FACS. qPCR analysis of three transcriptional variants as well as the total C9orf72 transcripts showed an increase after targeted methylation of G4C2 repeats (Figure 2B and 2C). We also performed Pyro-seq to examine the methylation status of C9orf72 locus. The result in Figure 2D showed a comparable methylation level of CGI-1, suggesting a specific methylation is confined to the G4C2 repeat expansion, but not non-targeted CGI-1 region. Importantly, the percentage of RNA foci-positive cells were reduced from 4-6% in unedited cells to less than 1% in editing in Figure 2E, suggesting methylation of G4C2 will decrease the formation of RNA foci, one of the molecular hallmarks leading to neurodegeneration. To evaluate the rescue effect of targeted methylation of G4C2 repeats, we have successfully established a robust motor neuron differentiation protocol. As shown in Figure 2F, motor neurons derived from C9orf72-ALS/FTD patient iPSC and isogenic control expressed Isl1/2 and HB9, two cell type specific marks to confirm the motor neuron identity. We are generating motor neurons and cortical neurons from methylation edited iPSCs, and then performing a similar set of characterization experiments. In addition, we also imported two C9-ALS/FTD mouse models to prepare for the DNA methylation editing in vivo. Conclusion We have generated/obtained four isogenic pairs of C9orf72 ALS/FTD iPSCs with stringent control lacking G4C2 repeats. We have established isoform-specific qPCR assay allowing for the measurement of three C9ofr72 isoforms expression as well as the total C9orf72 transcripts, RNA- FISH method to examine the RNA foci containing G4C2 repeats, and immunofluorescence and blotting to examine the dipeptide repeat proteins. Characterization of these iPSCs showed that the expression of C9orf72 transcript and protein are reduced. The molecular hallmark of RNA foci, but not DPRs, can be detected in these C9orf7-ALS/FTD iPSCs. We have performed DNA methylation editing of the G4C2 repeats in these C9orf72-ALS/FTD iPSCs with isogenic controls. Targeted DNA methylation of G4C2 repeats increases the expression of C9orf72 transcription. Importantly, we observed that targeted DNA methylation of the G4C2 repeats reduced the formation of RNA foci, one of the molecular hallmarks for C9-ALS/FTD pathology. We are examining the potential off-target effect at DNA methylation and transcription levels. We also successfully established a robust motor neuron differentiation protocol. We are differentiating these edited C9orf72-ALS/FTD iPSCs into motor and cortical neurons to examine the rescue effect of DNA methylation editing on the neurodegeneration process. Summary • DNA methylation editing in C9-ALS iPSC by viral delivery. • Examination of the DNA methylation status, RNA foci, and DPRs in edited C9-ALS iPSCs. • Characterization of the motor neurons derived from mock, isogenic control, and methylation edited C9-ALS iPSCs. • Characterization of the two C9-ALS mouse models in a time course experiment. • Editing the G4C2 repeat and CGIs in vivo by viral delivery. • Editing the G4C2 repeat and CGIs in crossed mouse model (C9-ALS;CAG-LSL-dCas9- Dnmt3/Tet). • Characterization of the edited C9-ALS mice. Example 2 As illustrated in Figures 3A and 3B, we carried out studies to demonstrate that our in vivo editing model can help ameliorate C9-ALS/FTD molecular phenotypes. Thus, we bred mice expressing a BAC carrying the human C9orf72 gene with the hexanucleotide repeat expansion (HRE) with mice carrying the CRISPR/dCas9-DNMT3A-P2A-GFP tool with a GFP reporter. In the early neonatal window, we delivered a guide RNA with mCherry marker targeting either CGI- 1, G4C2, or CGI-2 to offspring of this cross (Figure 3C). We aged these mice for 1 month and harvested their brains for sense strand RNA foci FISH (Figure 3D). Quantifying foci in the brainstem and cortex, we observed a relative drop in percentage foci-positive cells compared to scramble-injected controls for all three targeted regions, with the G4C2-targeted individual showing near baseline levels (Figure 3E). These results were reflected when assaying antisense transcript foci for G4C2 and CGI-2 sgRNAs, though interestingly not the CGI-1 sgRNA. These data suggest hypermethylating CGI-1, the HRE, or CGI-2 can help ameliorate C9-ALS/FTD molecular phenotype. METHODS Plasmid design and construction PCR amplified Tet1 catalytic domain from pJFA344C7 (Addgene plasmid: 49236), Tet1 inactive catalytic domain from MLM3739 (Addgene plasmid: 49959), and tagBFP (synthesized gene block) were cloned into FUW vector (Addgene plasmid: 14882) with AscI, EcoRI and PfIMI to package lentiviruses. The target sgRNA expression plasmids were cloned by inserting annealed oligos into modified pgRNA plasmid (Addgene plasmid: 44248) with AarI site. A synthetic gBlock encoding the bacteriophage AcrIIA4 purchased from IDT was cloned into a modified FUW vector with AscI and EcoRI to package lentiviruses. All constructs were sequenced before transfection. Cell culture and lentivirus production iPSCs were cultured either with mTeSR1 medium (STEMCELL, #85850) or on irradiated mouse embryonic fibroblasts (MEFs) with standard hESCs medium: [DMEM/F12 (Invitrogen) supplemented with 15% fetal bovine serum (GIBCO HI FBS, 10082-147), 5% KnockOut Serum Replacement (Invitrogen), 2 mM L-glutamine (MPBio), 1% nonessential amino acids (Invitrogen), 1% penicillin-streptomycin (Lonza), 0.1 mM b-mercaptoethanol (Sigma) and 4 ng/ml FGF2 (R&D systems)]. Lentiviruses expressing dCas9-Tet1-P2A-BFP, sgRNAs, and AcrIIA4 were produced by transfecting HEK293T cells with FUW constructs or pgRNA constructs together with standard packaging vectors (pCMV-dR8.74 and pCMV-VSVG) followed by ultra-centrifugation-based concentration. Virus titer (T) was calculated based on the infection efficiency for 293T cells, where T = (P*N) / (V), T = titer (TU/ul), p = % of infection positive cells according to the fluorescence marker, N = number of cells at the time of transduction, V = total volume of virus used. Note TU stands for transduction unit. Lentiviruses labeling NPCs (EF1A-GFP and EF1A-RFP) were purchased from Cellomics Technology. Multi-electrode array recording Two- or four-week-old differentiating neuronal cultures were dissociated using Accutase and 5 X 105 cells were plated on each single well in the PEI-coated Axion Biosystems # M768- GL1-30Pt200 arrays. Recordings of spontaneous activities during a 5-minute period were performed on days. Biological triplicates for each type of neurons were included. Immunocytochemistry, immunohistochemistry, microscopy, and image analysis iPSCs and neurons were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Cells were permeabilized with PBST (1 x PBS solution with 0.1% Triton X-100) before blocking with 10% Normal Donkey Serum (NDS) in PBST. Cells were then incubated with appropriately diluted primary antibodies in PBST with 5% NDS for 1 hours at room temperature or 12 hours at 4℃, washed with PBST for 3 times at room temperature and then incubated with desired secondary antibodies in TBST with 5% NDS and DAPI to counter stain the nuclei. The following antibodies were used in this study: Chicken anti-GFP (1:1000, Aves Labs), Rabbit anti-FMRP (1:50, Cell Signaling), Chicken anti-MAP2 (1:1000, Encor Biotech), Goat anti-mCherry (1:1000, SICGEN). Images were captured on a Zeiss LSM710 confocal microscope and processed with Zen software, ImageJ/Fiji, and Adobe Photoshop. For imaging- based quantification, unless otherwise specified, 3-5 representative images were quantified and data were plotted as mean ± SD with Excel or Graphpad Prism. FACS analysis To isolate the infection-positive cell after lentiviral transduction, the treated cells were dissociated with trypsin and single-cell suspensions were prepared in growth medium subject to a BD FACSAria cell sorter according to the manufacture’s protocol. Data were analyzed with FlowJo software. Western blot Cells were lysed by RIPA buffer with proteinase inhibitor (Invitrogen), and subject to standard immunoblotting analysis. Mouse anti-Cas9 (1:1000, Active Motif), mouse a-Tubulin (1:1000, Sigma), mouse anti-FMR1polyG (1:1000, EMD Millipore), rabbit anti-FMRP (1:100, Cell Signaling) antibodies were used. RT-qPCR Cells were harvested using Trizol followed by Direct-zol (Zymo Research), according to manufacturer’s instructions. RNA was converted to cDNA using First-strand cDNA synthesis (Invitrogen SuperScript III). Quantitative PCR reactions were prepared with SYBR Green (Invitrogen), and performed in 7900HT Fast ABI instrument. Bisulfite Conversion, PCR and Sequencing Bisulfite conversion of DNA was established using the EpiTect Bisulfite Kit (QIAGEN) following the manufacturer’s instructions. The resulting modified DNA was amplified by first round of nested PCR, following a second round using loci specific PCR primers. The first round of nested PCR was done as follows: 94℃ for 4 min; 55℃ for 2 min; 72℃ for 2 min; Repeat steps 1-31 X; 94℃ for 1 min; 55℃ for 2 min; 72℃ for 2 min; Repeat steps 5-735X; 72℃ for 5 min; Hold 12℃. The second round of PCR was as follows: 95℃ for 4 min; 94℃ for 1 min; 55℃ for 2 min; 72℃ for 2 min; Repeat steps 2-435 X; 72℃ for 5 min; Hold 12℃. The resulting amplified products were gel-purified, sub-cloned into a pCR2.1-TOPO-TA cloning vector (Life technologies), and sequenced. DNA Methylation analysis Pyro-seq of all bisulfite converted genomic DNA samples were performed with PyroMark Q48 Autoprep (QIAGEN) according to the manufacturer’s instructions. Methylation analysis of CGG trinucleotide repeats: Methylation status of CGG repeats were analyzed by Claritas Genomics Inc. with Asuragen AmplideX_ mPCR approach. The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. While certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in the foregoing description is offered by way of illustration only and not as a limitation.

Claims

What is claimed is: 1. A method of treating amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) in a subject, the method comprising administering to the subject a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase)-dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.
2. The method of claim 1, wherein the DNase-dead CRISPR-Cas nuclease is dCas9 or dCpf1.
3. The method of claims 1 or 2, wherein the effector domain is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) methylcytosine dioxygenase protein.
4. The method of claim 3, wherein the TET protein is Tet1.
5. The system of claim 3, wherein the DNMT protein is Dnmt3a.
6. The method of claims 1 or 2, wherein the effector domain is TET1 or Dnmt3a.
7. The method of any preceding claim, wherein the effector domain has an activity to modify an epigenome.
8. The method of any preceding claim, wherein the effector domain is an enzyme that modifies methylation state of DNA.
9. The method of any preceding claim, wherein the first polynucleotide sequence and the second polynucleotide sequence are on a single vector or on different vectors.
10. The method of any preceding claim, wherein the one or more target sequences are in the C9orf72 gene.
11. The method of any preceding claim, wherein the one or more target sequences are in GGGGCC (G4C2) repeats in the C9orf72 gene and/or a C9orf72 promoter region.
12. The method of any preceding claim, wherein the system modifies methylation state of the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region.
13. The method of any preceding claim, wherein the one or more guide sequences is/are one or more CRISPR RNA (crRNA) molecules, one or more single-guide RNA (sgRNA) molecules, one or more guide RNA (gRNA) molecules, or combinations thereof.
14. The method of any preceding claim, wherein the second polynucleotide sequence encodes two or more crRNA molecules that hybridize to two or more target sequences.
15. A method for modifying an epigenome of a cell, the method comprising contacting the cell with a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase) dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences in the C9orf72 gene.
16. The method of claim 15, wherein the one or more target sequences are in GGGGCC (G4C2) repeats in the C9orf72 gene and/or a C9orf72 promoter region.
17. The method of any of claims 15 or 16, wherein the system modifies methylation state of the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region.
18. The method of any of claims 15-17, wherein the DNase-dead CRISPR-Cas nuclease is dCas9 or dCpf1.
19. The method of any of claims 15-18, wherein the effector domain is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) methylcytosine dioxygenase protein.
20. The method of any of claims 15-19, wherein the effector domain is TET1 or Dnmt3a.
21. The method of any of claims 15-20, wherein the cell is a neuron.
22. The method of any of claims 15-20, wherein the cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).
23. The method of claim 22, wherein the iPSC is derived from a fibroblast of a subject.
24. The method of claims 22 or 23, further comprising culturing the iPSC to differentiate into a neuron.
25. The method of claims 21 or 24, further comprising administering the neuron to a subject.
26. A method of ameliorating motor neuron degeneration in a subject, the method comprising administering to the subject a system comprising: (a) a first polynucleotide sequence encoding a fusion protein comprising a deoxyribonuclease (DNase)-dead CRISPR-Cas nuclease and an effector domain; and (b) a second polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences.
27. The method of claim 26, wherein the DNase-dead CRISPR-Cas nuclease is dCas9 or dCpf1.
28. The method of claims 26 or 27, wherein the effector domain is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) methylcytosine dioxygenase protein.
29. The method of any of claims 26-28, wherein the effector domain is TET1 or Dnmt3a.
30. The method of any of claims 26-29, wherein the one or more target sequences are in the C9orf72 gene.
31. The method of any of claims 26-30, wherein the one or more target sequences are in GGGGCC (G4C2) repeats in the C9orf72 gene and/or a C9orf72 promoter region.
32. The method of any of claims 26-31, wherein the system modifies methylation state of the G4C2 repeats in the C9orf72 gene and/or the C9orf72 promoter region.
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