Crispr-cas component systems, methods and compositions for sequence manipulation

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. 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 and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

What is claimed is: 
     
         1 . An engineered composition comprising:
 a Cas9 protein; and   a guide comprising:   (a) an engineered guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif,   (b) a tracr mate sequence capable of hybridizing to a tracr sequence, and   (c) the tracr sequence.   
     
     
         2 . An engineered composition comprising one or more vectors comprising one or more nucleotide sequences encoding a Cas9 protein, and one or more nucleotide sequences encoding a guide, the guide comprising:
 (a) an engineered guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif,   (b) a tracr mate sequence capable of hybridizing to a tracr sequence, and   (c) the tracr sequence.   
     
     
         3 . The composition according to  claim 1  or  2 , wherein the Cas9 protein is translated from mRNA. 
     
     
         4 . The composition according to  claim 1  or  2 , wherein the Cas9 protein is delivered in the form of a protein. 
     
     
         5 . The composition according to  claim 1  or  2 , wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation. 
     
     
         6 . The composition according to  claim 1  or  2 , wherein (b) and (c) are fused or covalently linked to one another with intervening nucleotides. 
     
     
         7 . The composition according to  claim 1  or  2 , wherein the Cas9 protein is derived from  Streptococcus  or  Staphylococcus.    
     
     
         8 . The composition according to  claim 7 , wherein the Cas9 protein is  Streptococcus pyogenes  Cas9. 
     
     
         9 . The composition according to  claim 1  or  2 , further comprising a template. 
     
     
         10 . The composition according to  claim 1  or  2 , wherein the Cas9 protein is linked or fused to one or more NLSs. 
     
     
         11 . The composition according to  claim 2 , wherein the nucleotide sequence encoding the Cas9 protein is codon optimized for expression in a eukaryotic cell. 
     
     
         12 . The composition according to  claim 6 , wherein the tracr sequence comprises at least 40 nucleotides in length. 
     
     
         13 . The composition according to  claim 1  or  2 , wherein the composition is configured as a delivery composition suitable for delivery by an electroporation method. 
     
     
         14 . The composition according to  claim 1  or  2 , wherein two or more different guide sequences are provided for multiplexed targeting. 
     
     
         15 . The composition according to  claim 1  or  2 , wherein the Cas9 protein is linked or fused to at least one functional domain. 
     
     
         16 . The composition according to  claim 15 , wherein the functional domain is a transcriptional activation domain or a transcriptional repression domain. 
     
     
         17 . The composition according to  claim 1  or  2 , wherein the engineered composition comprises or encodes two or more guides each capable of hybridizing to a different target sequence in the eukaryotic cell. 
     
     
         18 . The composition according to  claim 1  or  2 , wherein the engineered composition further comprises a recombination template. 
     
     
         19 . The composition of  claim 18 , wherein the recombination template is inserted by homology-directed repair (HDR). 
     
     
         20 . A method of modifying one or more target sequences comprising contacting a cell or population of cells with the composition of  claim 1  or  2 , wherein the guide sequence directs sequence-specific binding to a target sequence in the cell or population of cells, whereby the target sequence is modified. 
     
     
         21 . The method of  claim 20 , wherein the modification of the one or more target sequences results in:
 a. a cell or population of cells comprising altered expression levels of one or more gene products;   b. a cell or population of cells that produces and/or secrete an endogenous or non-endogenous biological product or chemical compound   
     
     
         22 . An engineered cell comprising one or more modified genes modified according to the method of  claim 20 . 
     
     
         23 . The engineered cell of  claim 22 , wherein the cell produces and/or secretes an endogenous or non-endogenous biological product or chemical compound. 
     
     
         24 . The engineered cell of  claim 23 , wherein the biological product is a protein or an RNA. 
     
     
         25 . A cell line comprising the engineered cell of  claim 22  and progeny thereof. 
     
     
         26 . A plant or animal model comprising the engineered cell of  claim 22  and progeny thereof. 
     
     
         27 . A composition comprising the engineered cell of  claim 22 . 
     
     
         28 . The composition of  claim 27  formulated for use as a therapeutic. 
     
     
         29 . A biological product or chemical compound produced by the engineered cell of  claim 22 . 
     
     
         30 . An engineered cell or progeny thereof, the cell being engineered by use of an engineered CRISPR-Cas complex. 
     
     
         31 . The cell or progeny thereof of  claim 30  that is isolated. 
     
     
         32 . The cell or progeny thereof of  claim 30  that is further used as a therapeutic. 
     
     
         33 . The cell or progeny thereof of  claim 30  from which a product is isolated. 
     
     
         34 . A product produced by the cell or progeny thereof of  claim 30 . 
     
     
         35 . The product of  claim 34 , wherein the product is a protein or an RNA. 
     
     
         36 . The product of  claim 35 , wherein the protein comprises a mutation. 
     
     
         37 . A pharmaceutical composition for treatment of a disease or disorder, comprising the cell or progeny thereof of  claim 30 . 
     
     
         38 . The pharmaceutical composition according to  claim 37 , wherein the treatment results in genetic changes in one or more cells. 
     
     
         39 . The pharmaceutical composition according to  claim 37 , wherein the treatment results in correction of one or more defective genotypes. 
     
     
         40 . The pharmaceutical composition according to  claim 37 , wherein the treatment results in improved phenotype. 
     
     
         41 . The cell or progeny thereof of  claim 30 , wherein the cell comprises a mutation in a protein expressed from a gene comprising the target sequence. 
     
     
         42 . The cell or progeny thereof of  claim 41 , wherein the cell comprises deletion of a genomic region comprising the target sequence. 
     
     
         43 . The cell or progeny thereof of  claim 41 , wherein the cell comprises integration of an exogenous sequence by homology-directed repair. 
     
     
         44 . The cell or progeny thereof of  claim 41 , wherein the cell comprises decreased transcription of a gene associated with the target sequence. 
     
     
         45 . The cell or progeny thereof of  claim 41 , wherein the cell comprises increased transcription of a gene associated with the target sequence. 
     
     
         46 . The product of  claim 34 , wherein the product is a mutated protein or product provided by a template.