ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCE MANIPULATION
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.
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 guide RNAs that hybridize with target sequences in polynucleotide loci in a eukaryotic cell, the guide RNA comprising a guide sequence, a tracr sequence, and a tracr mate sequence,
b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, said protein comprising a nuclear localization signal (NLS);
wherein components (a) and (b) are located on same or different vectors of the system,
wherein the tracr sequence is 30 or more nucleotides in length, and
whereby the one or more guide RNAs target the polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the polynucleotide loci, whereby sequence of the polynucleotide loci is modified; and, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together.
2 . An engineered, non-naturally occurring Type II CRISPR-Cas vector system according to claim 1 , wherein the Cas9 protein comprises one or more mutations in a catalytic domain, such that the mutated Cas9 protein lacks the ability to cleave one strand of the polynucleotide loci, and is a nickase.
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 claim 2 wherein the Cas9 protein comprises one or more mutations in the RuvC I, RuvC II or RuvC III catalytic domains.
6 . The system of claim 2 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 claim 1 or 2 , wherein the guide RNA is a chimeric RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence.
8 . The system of claim 7 wherein hybridization between the tracr sequence and the tracr mate sequence produces a transcript having a secondary structure.
9 . The system of claim 8 , wherein the secondary structure is a hairpin.
10 . The system of claim 1 or 2 , wherein the eukaryotic cell is a mammalian cell or a human cell.
11 . The system of claim 1 or 2 , wherein the nucleotide sequence encoding the Cas9 protein is codon optimized for expression in a eukaryotic cell.
12 . The system of claim 1 or 2 , wherein a repair template is inserted into the cleaved polynucleotide loci.