Delivery, use and therapeutic applications of the crispr-cas systems and compositions for genome editing

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 CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing 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 - 30 . (canceled) 
     
     
         31 . A composition comprising a CRISPR-Cas system for treating an ocular or auditory genetic disease, wherein the CRISPR-Cas system comprises:
 a CRISPR-Cas system RNA or a polynucleotide encoding the CRISPR-Cas system RNA, wherein the CRISPR-Cas system RNA comprises: (a) a guide sequence capable of hybridizing to a target sequence associated with the ocular or auditory genetic disease, (b) a tracr mate sequence, and (c) tracr sequence; and   a Cas9 or a polynucleotide encoding the Cas9;   wherein the composition is formulated for localized administration to a subject's eye or cochlea, and wherein when locally administered the CRISPR-Cas system is capable of forming a CRISPR-Cas complex comprising the Cas9 and the CRISPR-Cas system RNA.   
     
     
         32 . The composition according to  claim 31 , wherein the CRISPR-Cas system is a multiplexed CRISPR-Cas system comprising multiple guide sequences each capable of hybridizing to a different target sequence. 
     
     
         33 . The composition according to  claim 31 , wherein the Cas9 is fused to one or more nuclear localization signals (NLS). 
     
     
         34 . The composition according to  claim 31 , wherein the Cas9 is fused to two or more NLSs. 
     
     
         35 . The composition according to  claim 31 , wherein the Cas9 is a nuclease directing cleavage of both strands at the location of the target sequence. 
     
     
         36 . The composition according to  claim 31 , wherein the Cas9 is a nickase directing cleavage of a single strand at the location of the target sequence. 
     
     
         37 . The composition according to  claim 31 , wherein the Cas9 comprises one or more mutations in a catalytic domain, and wherein the Cas9 is fused to a heterologous functional domain. 
     
     
         38 . The composition according to  claim 37 , wherein the heterologous functional domain has one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. 
     
     
         39 . The composition according to  claim 31 , wherein the Cas9 is  S. aureus  Cas9. 
     
     
         40 . The composition according to  claim 31 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         41 . The composition according to  claim 31 , wherein the CRISPR-Cas system RNA is a chimeric RNA, and wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation. 
     
     
         42 . The composition according to  claim 31 , wherein the Cas9 is complexed with the CRISPR-Cas system RNA. 
     
     
         43 . The composition according to  claim 31 , wherein the polynucleotide encoding the Cas9 is an mRNA. 
     
     
         44 . The composition according to  claim 31 , wherein the CRISPR-Cas system is comprised in a lipid particle or a viral vector. 
     
     
         45 . The composition according to  claim 31 , further comprising a template polynucleotide for homology directed repair. 
     
     
         46 . The composition according to  claim 31 , wherein the subject is a mammalian or human subject. 
     
     
         47 . The composition according to  claim 31  wherein the ocular genetic disease is a retinal disease or a hereditary retinal disease. 
     
     
         48 . The composition according to  claim 31 , wherein the ocular genetic disease is Leber congenital amaurosis, Usher syndrome, Stargardt disease, Sorsby dystrophy, Cone-rod dystrophy, retinitis pigmentosa, or age-related macular degeneration. 
     
     
         49 . The composition according to  claim 31 , wherein the auditory genetic disease is a cochlear-cell associated disease, hearing impairment, deafness. 
     
     
         50 . The composition according to  claim 31 , which is formulated for single dose subretinal injection or intravitreal injection.