CRISPR having or associated with destabilization domains
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College
InventorsFeng Zhang, Bernd Zetsche, Amit Choudhary
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Abstract
The disclosure includes non-naturally occurring or engineered CRISPR Cas9, each associated with at least one destabilization domain (DD), along with compositions, systems and complexes involving the DD-CRISPR Cas9, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses therefor.
Claims29
A composition comprising:
- (a) a non-naturally occurring or engineered Cas9 attached to at least two destabilization domains (DD);
- wherein a first DD is attached to the N-terminus of the Cas9 and a second DD is attached to the C-terminus of the Cas9, the first and second DDs being the same or different;
- wherein the Cas9 is attached to the at least two DDs via fusion, tether, or non-covalent bond;
- (b) a guide RNA that forms a CRISPR complex with the Cas9;
- and
- (c) at least one stabilizing ligand that binds to at least one of the destabilization domains;
- wherein binding of the at least one stabilizing ligand to at least one of the destabilization domains inhibits degradation of the Cas9 by proteasome, and wherein the guide RNA directs sequence-specific binding of the CRISPR complex to a target DNA.
A composition comprising a non-naturally occurring or engineered Cas9 attached to at least two destabilization domains (DD);
- wherein a first DD is attached to the N-terminus of the Cas9 and a second DD is attached to the C-terminus of the Cas9, the first and second DDs being the same or different;
- wherein the Cas9 is attached to the at least two DDs via fusion, tether, or non-covalent bond;
- and wherein the Cas9 attached to the at least two DDs comprises an amino acid sequence selected from SEQ ID NOs:
- 56-61.
A composition comprising a non-naturally occurring or engineered split Cas9 attached to at least one destabilization domain (DD) via fusion, tether, or non-covalent bond, wherein the Cas9 is a Staphylococcus aureus Cas9 (SaCas9) split into an N-terminus portion (Cas9 (N)) and a C-terminus portion (Cas9 (C)).
A composition comprising a non-naturally occurring or engineered Cas9 attached to at least one destabilization domain (DD) via fusion, tether, or non-covalent bond, wherein the Cas9 is a Staphylococcus aureus Cas9 (SaCas9) having N580A mutation and is a nickase.
Dependencies are inferred from the claim text.