Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
First Claim
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1. A method of altering expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product comprising introducing into said eukaryotic cell an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
- a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, andb) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Staphylococcus aureus Cas9 protein,wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system,whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule;
the method further comprising inserting DNA into a cleaved strand of the DNA molecule;
whereby expression of the at least one gene product is altered; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together.
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Abstract
The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.
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Citations
30 Claims
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1. A method of altering expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product comprising introducing into said eukaryotic cell an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
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a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Staphylococcus aureus Cas9 protein, wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule; the method further comprising inserting DNA into a cleaved strand of the DNA molecule; whereby expression of the at least one gene product is altered; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A CRISPR-Cas system-mediated genome targeting method in a eukaryotic cell containing a DNA molecule having a target sequence comprising introducing into said eukaryotic cell an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
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a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Staphylococcus aureus Cas9 protein, wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)) and components (a) and (b) are located on same or different vectors of the system; the method further comprising inserting DNA into a cleaved strand of the DNA molecule; whereby there is CRISPR-Cas system-mediated genome targeting through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby there is genome editing; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 29)
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25. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a Staphylococcus aureus Cas9 protein, at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, one or more NLS(s), and DNA for insertion into a cleaved strand of the DNA molecule;
- wherein the Cas9 protein cleaves the DNA molecule, and the DNA for insertion inserts into a cleaved strand of the DNA molecule, whereby the CRISPR-Cas system when introduced into a eukaryotic cell having the DNA molecule provides mediated genome targeting; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together. - View Dependent Claims (26, 27, 28, 30)
- wherein the Cas9 protein cleaves the DNA molecule, and the DNA for insertion inserts into a cleaved strand of the DNA molecule, whereby the CRISPR-Cas system when introduced into a eukaryotic cell having the DNA molecule provides mediated genome targeting; and
Specification