Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

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.

What is claimed is: 
     
         1 . A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
 a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and   b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,   wherein components (a) and (b) are located on same or different vectors of the system,   whereby the guide RNA targets the target sequence and the Cas9 protein generates at least one site specific break in the DNA molecule, wherein the at least one site specific break is repaired through a cellular repair mechanism; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.   
     
     
         2 . The method of  claim 1 , wherein the cellular repair mechanism is Non-homologous end joining (NHEJ). 
     
     
         3 . The method of  claim 1 , wherein the cellular repair mechanism is Homology-directed repair (HDR). 
     
     
         4 . The method of  claim 1 , wherein the Cas9 protein is a nickase that generates a site specific break in only one strand of the DNA molecule. 
     
     
         5 . The method of  claim 1 , wherein the method further comprises the insertion of a recombination template into the site specific break in the DNA molecule. 
     
     
         6 . The method of  claim 1 , wherein the expression of two or more gene products is altered. 
     
     
         7 . The method of  claim 1 , wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)). 
     
     
         8 . The method of  claim 1 , wherein the CRISPR-Cas system comprises a trans-activating cr (tracr) sequence. 
     
     
         9 . The method of  claim 1 , wherein the guide RNAs comprise a guide sequence fused to a tracr sequence. 
     
     
         10 . The method of  claim 1 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell. 
     
     
         11 . The method of  claim 1 , wherein the eukaryotic cell is a mammalian or human cell. 
     
     
         12 . The method of  claim 1 , wherein the expression of one or more gene products is increased. 
     
     
         13 . The method of  claim 1 , wherein the expression of one or more gene products is decreased. 
     
     
         14 . The method of  claim 1 , wherein the one or more vectors are viral vectors. 
     
     
         15 . The method of  claim 1 , wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. 
     
     
         16 . A CRISPR-Cas system-mediated genome editing method comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding at least one gene product an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
 a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and   b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,   wherein components (a) and (b) are located on same or different vectors of the system,   whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby the Cas9 protein generates at least one site specific break in the DNA molecule, whereby there is genome editing, and, wherein the Cas9 protein and the guide RNA do not naturally occur together.   
     
     
         17 . The method of  claim 16 , wherein the at least one site specific break is repaired through a NHEJ cellular repair mechanism. 
     
     
         18 . The method of  claim 16 , wherein the at least one site specific break is repaired through a HDR cellular repair mechanism. 
     
     
         19 . The method of  claim 16 , wherein the Cas9 protein is a nickase that generates a site specific break in only one strand of the DNA molecule. 
     
     
         20 . The method of  claim 16 , wherein the method further comprises the insertion of a recombination template into the site specific break in the DNA molecule. 
     
     
         21 . The method of  claim 16 , wherein the expression of two or more gene products is altered. 
     
     
         22 . The method of  claim 16 , wherein the CRISPR-Cas system further comprises one or more NLS(s). 
     
     
         23 . The method of  claim 16 , wherein the CRISPR-Cas system comprises a tracr sequence. 
     
     
         24 . The method of  claim 16 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell. 
     
     
         25 . The method of  claim 16 , wherein the eukaryotic cell is a mammalian or human cell. 
     
     
         26 . The method of  claim 16 , wherein the expression of one or more gene products is increased. 
     
     
         27 . The method of  claim 16 , wherein the expression of one or more gene products is decreased. 
     
     
         28 . An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product and the Cas9 protein generates at least one site specific break in the DNA molecule, wherein the at least one site specific break is repaired through a cellular repair mechanism; and, wherein the Cas9 protein and the guide RNA do not naturally occur together. 
     
     
         29 . The CRISPR-Cas system of  claim 28 , wherein the cellular repair mechanism is NHEJ. 
     
     
         30 . The CRISPR-Cas system of  claim 28 , wherein the cellular repair mechanism is HDR.