Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
First Claim
1. A non-naturally occurring or engineered composition for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product;
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,said 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 fusion of a Type-II Cas9 protein and one or more protein domains,wherein;
components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together,whereby expression of the at least one gene product is altered through the CRISP R-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, and whereinthe one or more protein domains comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity.
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Accused Products
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 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 with additional functional domains. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.
376 Citations
43 Claims
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1. A non-naturally occurring or engineered composition for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product;
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said 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, and b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, wherein; components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together, whereby expression of the at least one gene product is altered through the CRISP R-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, and wherein the one or more protein domains comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
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2. A multiplexed non-naturally occurring or engineered composition for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product;
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said 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, and b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, wherein; components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together, whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISP R-Cas system and PAM recognition, and wherein the one or more protein domains comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity, and the system includes at least two guide RNAs, each of which hybridizes with a different target sequence whereby the system is multiplexed.
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
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3. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product;
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising; a) a first regulatory element operable in a eukaryotic cell operably linked to a guide sequence capable of hybridizing to a target sequence in the eukaryotic cell, and at least one or more tracr mate sequences, and b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, and c) a third regulatory element operably linked to a tracr sequence, wherein; components (a), (b) and (c) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together, whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISP R-Cas system and PAM recognition, and wherein the one or more protein domain comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity.
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
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4. A multiplexed CRISPR enzyme system composition comprising a vector system comprising one or more vectors for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product;
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising; a) a first regulatory element operable in a eukaryotic cell operably linked to a guide sequence capable of hybridizing to a target sequence in the eukaryotic cell, and at least one or more tract mate sequences, b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, and c) a third regulatory element operably linked to a tracr sequence, wherein; components (a), (b) and (c) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together, whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, and wherein the one or more protein domain comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity, and the system comprises at least two different guide sequences, each capable of hybridizing to a different target sequence, whereby the system is multiplexed.
- said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
Specification