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 . An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising:
 a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system polynucleotide sequences comprising a guide sequence, a tracr RNA, and a tracr mate sequence, wherein the guide sequence hybridizes with one or more target sequences in polynucleotide loci in a eukaryotic cell,   b) a second regulatory element 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,   wherein the CRISPR-Cas system comprises two or more nuclear localization signals (NLSs) expressed with the nucleotide sequence encoding the Cas9 protein,   whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide loci, whereby the sequence of the one or more polynucleotide loci is modified.   
     
     
         2 . An engineered, non-naturally occurring Type II CRISPR-Cas vector system according to  claim 1 ,
 wherein the Cas9 protein is mutated with respect to a corresponding wild type Cas9 protein such that the mutated protein is a nickase that lacks the ability to cleave one strand of a target polynucleotide,   whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves only one strand of the polynucleotide loci, whereby the sequence of the one or more polynucleotide loci is modified.   
     
     
         3 . The system of  claim 1  or  2 , wherein the vectors are viral vectors. 
     
     
         4 . The system of  claim 3 , wherein the viral vectors are retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors. 
     
     
         5 . The system of any of  claims 2 - 4  wherein the Cas9 protein comprises one or more mutations in the RuvC I, RuvC II or RuvC III catalytic domains. 
     
     
         6 . The system of any of  claims 2 - 4  wherein the Cas9 protein comprises a mutation selected from the group consisting of D10A, H840A, N854A and N863A with reference to the position numbering of a  Streptococcus pyogenes  Cas9 (SpCas9) protein. 
     
     
         7 . The system of any preceding claim, wherein 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. 
     
     
         8 . The system of  claim 7 , 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. 
     
     
         9 . The system of any preceding claim, wherein the one or more CRISPR-Cas system polynucleotide sequences comprise a guide sequence fused to a trans-activating cr (tracr) sequence. 
     
     
         10 . The system of any preceding claim, wherein the CRISPR-Cas system polynucleotide sequence is a chimeric RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence. 
     
     
         11 . The system of any preceding claim, wherein the eukaryotic cell is a mammalian cell or a human cell. 
     
     
         12 . The system of any preceding claim, wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell. 
     
     
         13 . Use of the system of any of  claims 1  to  12  for genome engineering, provided the use does not comprise a process for modifying the germ line genetic identity of human beings, and provided that said use is not a method for treatment of the human or animal body by surgery or therapy. 
     
     
         14 . The use of  claim 13  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. 
     
     
         15 . The use of  claim 13  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. 
     
     
         16 . The use of  claim 13  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. 
     
     
         17 . The use of  claim 15  or  16  wherein the inserting is by homologous recombination. 
     
     
         18 . Use of the system of any of  claims 1  to  12  in the production of a non-human transgenic animal or transgenic plant.