CRISPR-Cas component systems, methods and compositions for sequence manipulation
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorFeng Zhang
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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.
Claims41
A CRISPR-Cas complex-mediated method for the production of a multicellular genetically modified non-human animal or multicellular genetically modified plant, the method comprising introducing into a cell of the multicellular non-human animal or plant containing one or more target sequences, wherein the target sequence is adjacent to a Protospacer Adjacent Motif (PAM), an engineered, non-naturally occurring 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 complex guide RNAs that hybridize with the target sequence(s), and
- 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, and
- wherein the guide RNA comprises a trans-activating cr (tracr) sequence which is 30 or more nucleotides in length,
- whereby when transcribed, the one or more guide RNAs form the CRISPR-Cas complex with the Cas9 protein, and the one or more guide RNAs direct sequence-specific binding of the CRISPR complex to one or more target sequences, and there is PAM recognition, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together, and
- whereby the multicellular genetically modified non-human animal or multicellular genetically modified plant is produced, and displays a phenotype or carries DNA to display a phenotype of the genetic modification.
A CRISPR-Cas complex-mediated method for the production of a multicellular genetically modified non-human animal or multicellular genetically modified plant, the method comprising delivery to one or more target sequences in a cell of the multicellular non-human animal or plant of:
- a Cas9 protein;
- a guide sequence linked to a tracr mate sequence;
- and
- a tracr sequence;
- wherein the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence in the cell, whereby the multicellular genetically modified non-human animal or multicellular genetically modified plant is produced, and displays a phenotype or carries DNA to display a phenotype of the genetic modification.
A CRISPR-Cas complex-mediated method for the production of a multicellular genetically modified non-human animal or multicellular genetically modified plant, the method comprising delivery to a cell of the multicellular non-human animal or plant having one or more target sequences of a Cas9 protein, or a nucleic acid molecule encoding the Cas9 protein;
- and a guide sequence linked to a tracr mate sequence;
- and a tracr sequence, or one or more nucleic acid molecules encoding the guide sequence linked to the tracr mate sequence and the tracr sequence,
- wherein the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence in the cell, whereby the multicellular genetically modified non-human animal or multicellular genetically modified plant is produced, and displays a phenotype or carries DNA to display a phenotype of the genetic modification.
Dependencies are inferred from the claim text.