Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

1 - 40 . (canceled) 
     
     
         41 . An engineered CRISPR-Cas system for in vivo genome editing in a multicellular organism, comprising:
 a Cas9 protein or a polynucleotide encoding the Cas9 protein, wherein the Cas9 protein is fused to at least one nuclear localization signal (NLS);   a first CRISPR-Cas system chimeric RNA engineered to hybridize to a first target sequence, or a polynucleotide encoding the first CRISPR-Cas system chimeric RNA, wherein the first chimeric RNA is capable of forming a first CRISPR complex with the Cas9 protein and directing sequence-specific binding of the first CRISPR complex to the first target sequence in the eukaryotic cell;   a second CRISPR-Cas system chimeric RNA engineered to hybridize to a second target sequence, or a polynucleotide encoding the second CRISPR-Cas system chimeric RNA, wherein the second chimeric RNA is capable of forming a second CRISPR complex with the Cas9 protein and directing sequence-specific binding of the second CRISPR complex to the second target sequence in the eukaryotic cell, wherein the first target sequence and second target sequence flank a genomic locus of interest to thereby mediate excision of the genomic locus of interest by the first and second CRISPR complexes in the eukaryotic cell, wherein the excision of the genomic locus of interest produces a phenotypic change in the multicellular organism; and   a third CRISPR-Cas system chimeric RNA engineered to hybridize to a third target sequence, or a polynucleotide encoding the third CRISPR-Cas system chimeric RNA, wherein the third chimeric RNA is capable of forming a third CRISPR complex with the Cas9 protein and directing sequence-specific binding of the third CRISPR complex to the third target sequence in the eukaryotic cell, wherein the third target sequence is present in the polynucleotide encoding the Cas9 protein to thereby mediate self-inactivation of the Cas9 protein.   
     
     
         42 . The engineered CRISPR-Cas system of  claim 41 , wherein the Cas9 protein is fused to at least two NLSs. 
     
     
         43 . The engineered CRISPR-Cas system of  claim 41 , wherein the Cas9 protein is  S. pyogenes  Cas9. 
     
     
         44 . The engineered CRISPR-Cas system of  claim 41 , wherein the Cas9 protein is  S. aureus  Cas9. 
     
     
         45 . The engineered CRISPR-Cas system of  claim 41 , wherein the polynucleotide encoding the Cas9 is codon optimized for expression in a mammalian cell. 
     
     
         46 . The engineered CRISPR-Cas system of  claim 41 , wherein the polynucleotide encoding the Cas9 is delivered in a vector. 
     
     
         47 . The engineered CRISPR-Cas system of  claim 41 , wherein the polynucleotide encoding the Cas9 is delivered in a viral vector. 
     
     
         48 . The engineered CRISPR-Cas system of  claim 41 , wherein the polynucleotide encoding the Cas9 is delivered in an AAV or lentiviral vector. 
     
     
         49 . The engineered CRISPR-Cas system of  claim 41 , wherein the polynucleotide encoding the Cas9 is delivered in a liposome, a nanoparticle, an exosome, or a microvesicle. 
     
     
         50 . The engineered CRISPR-Cas system of  claim 41 , further comprising a repair template for homology-directed repair. 
     
     
         51 . The engineered CRISPR-Cas system of  claim 50 , wherein the repair template comprises at least 1,000 nucleotides in length for replacing the excised genomic locus of interest. 
     
     
         52 . The engineered CRISPR-Cas system of  claim 41 , wherein the Cas9 protein comprises at least one mutation in a catalytic domain and is a nickase. 
     
     
         53 . The engineered CRISPR-Cas system of  claim 52 , wherein the Cas9 protein comprises at least one mutation which is D10A, E762A, H840A, N854A, N863A, or D986A. 
     
     
         54 . The engineered CRISPR-Cas system of  claim 41 , wherein the Cas9 protein is fused to at least one heterologous protein domain. 
     
     
         55 . The engineered CRISPR-Cas system of  claim 54 , wherein the heterologous protein domain is a methylase, a demethylase, a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, or a cryptochrome. 
     
     
         56 . The engineered CRISPR-Cas system of  claim 41 , wherein the multicellular organism is a mammalian organism. 
     
     
         57 . The engineered CRISPR-Cas system of  claim 56 , wherein the eukaryotic cell is a brain cell, a neuronal cell, or a central nervous tissue cell. 
     
     
         58 . The engineered CRISPR-Cas system of  claim 41 , wherein the genomic locus of interest comprises a defective nucleotide element which is: a trinucleotide repeat comprising CTG, CAG, CGG, CCG, GAA or TTC, a tetranucleotide repeat comprising CCTG, a pentanucleotide repeat comprising ATTCT or AGAAT, a hexanucleotide repeat comprising GGGGCC, or a dodecanucleotide repeat comprising CCCCGCCCCGCG (SEQ ID NO: 1) or CGCGGGGCGGGG (SEQ ID NO: 2). 
     
     
         59 . The engineered CRISPR-Cas system of  claim 58 , wherein the defective nucleotide element is a trinucleotide repeat comprising CAG or CTG. 
     
     
         60 . The engineered CRISPR-Cas system of  claim 58 , wherein the defective nucleotide element is associated with a brain or central nervous system disease or disorder which is: Fragile X (FXS), Spinocerebellar ataxia type-12 (SCA12), Friedreich Ataxia; Myotonic Dystrophy type-1 (DM1), Spinocerebellar ataxia type-8 (SCAB), Spinocerebellar ataxia type-10 (SCA10), Spinocerebellar ataxia type-31 (SCA31), Spinocerebellar ataxia type-1 (SCA1), Spinocerebellar ataxia type-2 (SCA2), Spinocerebellar ataxia type-3 (SCA3), Spinocerebellar ataxia type-6 (SCA6), Spinocerebellar ataxia type-7 (SCAT), Spinocerebellar ataxia type-17 (SCA17), Huntington's Disease (HD), Fragile X Tremor Ataxia (FXTAS), Unverricht-Lundborg disease (EPM1), Amyotrophic Lateral Sclerosis (ALS), Fronto Temporal Dementia (FTD), Myotonic Dystrophy type-2 (DM2), Oculopharyngeal muscular dystrophy (OPMD), Dentatorubral-pallidoluysian atrophy (DRPLA), Spinobulbar muscular atrophy (SBMA), or Huntington's disease like type-2 (HDL2).