Engineering of systems, methods and optimized guide 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 . A non-naturally occurring or engineered composition comprising:
 a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises   (a) a guide sequence of between 10-30 nucleotides in length, capable of hybridizing to a target sequence in a eukaryotic cell,   (b) a tracr mate sequence, and   (c) a tracrRNA sequence   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein when transcribed, the tracr mate sequence hybridizes to the tracrRNA sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,   wherein the CRISPR complex comprises a Type II Cas9 protein complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracrRNA sequence,   wherein the tracrRNA sequence is 50 or more nucleotides in length.   
     
     
         2 . A Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising
 I. a first regulatory element operably linked to a nucleotide sequence encoding a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence as defined in  claim 1 , and   II. a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein comprising one or more nuclear localization sequences, of sufficient strength to drive accumulation of said Cas9 protein in a detectable amount in the nucleus of a eukaryotic cell;   wherein components I and II are located on the same or different vectors of the system.   
     
     
         3 . The composition of  claim 1 , wherein the Cas9 enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said Cas9 enzyme in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         4 . The composition of  claim 1 , or the vector system of  claim 2 , wherein the Cas9 protein is a nuclease directing cleavage of both strands of the polynucleotide locus. 
     
     
         5 . The composition of  claim 1 , or the vector system of  claim 2 , wherein the Cas9 protein comprises one or more mutations in a catalytic domain, and is a nickase that cleaves only one strand of the polynucleotide locus. 
     
     
         6 . The vector system of  claim 2 , or any claim dependent thereon, wherein the nucleotide sequence encoding the Cas9 protein is codon-optimized for expression in a eukaryotic cell. 
     
     
         7 . The vector system of  claim 2 , or any claim dependent thereon, wherein the vectors are viral vectors. 
     
     
         8 . The vector system of  claim 7 , wherein the viral vectors are retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors. 
     
     
         9 . The vector system of  claim 2 , or any claim dependent thereon, wherein the vector system comprises nucleotide sequence(s) coding for two or more nuclear localisation signals (NLSs) expressed with the nucleotide sequence encoding the Cas9 protein. 
     
     
         10 . The vector system of  claim 9 , wherein when expressed at least one NLS is at or near amino-terminus of the Cas9 protein and/or at least one NLS is at or near carboxy terminus of the Cas9 protein. 
     
     
         11 . The vector system of  claim 9 , wherein at least one NLS is at or near amino-terminus of the Cas9 protein and at least one NLS is at or near carboxy terminus of the Cas9 protein. 
     
     
         12 . Use of the composition of  claim 1 , or the vector system of  claim 2  or any claim dependent thereon for genome engineering, provided that said use is not a method for treatment of the human or animal body by surgery or therapy, and provided that said use is not a process for modifying the germ line genetic identity of human beings. 
     
     
         13 . The use of  claim 12  wherein the genome engineering comprises modifying a target polynucleotide in a eukaryotic cell, modifying expression of a polynucleotide in a eukaryotic cell, generating a model eukaryotic cell comprising a mutated disease gene, or knocking out a gene. 
     
     
         14 . The use of  claim 12  wherein the use further comprises repairing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. 
     
     
         15 . The use of  claim 12  wherein the use further comprises editing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said edit results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. 
     
     
         16 . The use of  claim 13  or  14  wherein the inserting is by homologous recombination. 
     
     
         17 . Use of the composition of  claim 1 , or the vector system of  claim 2  or any claim dependent thereon, in the production of a non-human transgenic animal or transgenic plant.