CRISPR-CAS COMPONENT SYSTEMS, METHODS AND 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 modified eukaryotic cell or progeny thereof, wherein the modified eukaryotic cell is obtained ex vivo by treating a corresponding wild-type cell with a CRISPR-Cas system comprising (a) a Cas9 protein or a polynucleotide encoding the Cas9 protein and (b) a Cas9 guide targeting a genomic sequence in the corresponding wild-type cell positioned 5′ of a protospacer adjacent motif (PAM) recognized by the Cas9 protein, or a polynucleotide encoding the Cas9 guide,
wherein a CRISPR complex formed by the Cas9 protein and the Cas9 guide binds to and cleaves the genomic sequence, and wherein the modified eukaryotic cell comprises an insertion, deletion, or substitution of one or more nucleotides in the genomic sequence compared to the corresponding wild-type cell.
2 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the corresponding wild-type cell with a CRISPR-Cas system comprising the Cas9 guide and the Cas9 protein.
3 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the corresponding wild-type cell with a CRISPR-Cas system comprising a complex formed by the Cas9 guide and the Cas9 protein.
4 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the corresponding wild-type cell with a vector encoding the Cas9 guide and the Cas9 protein.
5 . The modified eukaryotic cell or progeny thereof of claim 4 , wherein the vector is a viral vector.
6 . The modified eukaryotic cell or progeny thereof of claim 5 , wherein the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.
7 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the corresponding wild-type cell with a CRISPR-Cas system comprising the Cas9 guide and an mRNA encoding the Cas9 protein.
8 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the CRISPR-Cas system further comprises a template DNA for homology-directed repair of the cleavage.
9 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the CRISPR-Cas system is delivered into the corresponding wild-type cell by microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.
10 . The modified eukaryotic cell or progeny thereof of claim 1 , which is a human cell.
11 . The modified eukaryotic cell or progeny thereof of claim 10 , which is a blood cell or a lymphocyte.
12 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the genomic sequence is associated with a genetic disease or disorder.
13 . The modified eukaryotic cell or progeny thereof of claim 12 , wherein the genetic disease or disorder is a blood disease or disorder.
14 . The modified eukaryotic cell or progeny thereof of claim 12 , wherein the genetic disease or disorder is sickle cell anemia or beta-thalassemia.
15 . The modified eukaryotic cell or progeny thereof of claim 12 , wherein the genetic disease or disorder is a cancer.
16 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell or progeny thereof has altered expression of one or more of PDCD1, CTLA4, B2M, and TCRA, compared to the corresponding wild-type cell.
17 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cas9 protein is from Streptococcus or Staphylococcus.
18 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cas9 protein is S. pyogenes Cas9 and the PAM is NGG; or wherein the Cas9 protein is S. thermophilus Cas9 and the PAM is NNAGAAW.
19 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cas9 guide is a chimeric RNA comprising, from 5′ to 3′, a guide sequence, a tracr-mate sequence and a tracr sequence covalently linked together.
20 . The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cas9 guide comprises a first RNA and a second RNA, wherein the first RNA comprises a guide sequence covalently linked to a tracr-mate sequence and the second RNA comprises a tracr sequence.
21 . A pharmaceutical composition comprising the modified eukaryotic cell or progeny thereof of claim 1 .