CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS
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.
1 . A composition comprising a non-naturally occurring or engineered CRISPR Cas9 associated with or fused to at least one destabilization domain (DD)
2 . The composition of claim 1 , wherein the Cas9 comprises an Sp Cas9, an Sa Cas9, an St Cas9, or an Fn Cas9.
3 . The composition of claim 1 or 2 , wherein the Cas9 comprises a Rec2 or HD2 truncation.
4 . The composition of claim 3 , wherein the truncation comprises removal or replacement with a linker.
5 . The composition of claim 4 , wherein the linker comprises a branch or otherwise allows for tethering of the DD and/or a functional domain.
6 . The composition of claim 1 , wherein the DD is associated with the CRISPR Cas9 by fusion with said CRISPR Cas9.
7 . The composition of claim 6 , which comprises at least one DD fused to the N-terminus or C-terminus of the CRISPR Cas9.
8 . The composition of claim 7 , comprising at least two DDs and wherein a first DD is fused to the N-terminus of the CRISPR Cas9 and a second DD is fused to the C-terminus of the CRISPR Cas9, the first and second DDs being the same or different.
9 . The composition of claim 6 , wherein the fusion comprises a linker between the DD and the CRISPR Cas9.
10 . The composition of claim 9 , wherein the linker comprises, consists essentially of or consists of: GlySer linker; or a localization signal.
11 . The composition of claim 1 , further comprising at least one Nuclear Export Signal (NES) or at least one Nuclear Localization Signal (NLS).
12 . The composition of claim 11 , wherein the Cas9 comprises two or more NESs.
13 . The composition of claim 7 , wherein one of the at least one DD comprises ER50 or DHFR50.
14 . The composition of claim 1 , wherein the Cas9 comprises at least one mutation.
15 . The composition of claim 14 , wherein the Cas9 comprises a nickase.
16 . The composition of claim 15 , wherein the Cas9 comprises Staphylococcus aureus Cas9 (SaCas9) and the mutation comprises N580A.
17 . The composition of claim 14 , wherein the Cas9 has substantially no nuclease activity due to the mutation(s).
18 . The composition of claim 1 , wherein the Cas9 comprises a split Cas9.
19 . The composition of claim 1 , wherein the Cas9 comprises a functional domain.
20 . A polynucleotide encoding the CRISPR Cas9 and associated or fused DD of claim 1 .
21 . The polynucleotide of claim 20 , wherein the encoded CRISPR Cas9 and associated DD are operably linked to a first regulatory element.
22 . The polynucleotide of claim 20 , wherein a DD is encoded and is operably linked to a second regulatory element.
23 . The polynucleotide of claim 21 , wherein the first regulatory element comprises a promoter and optionally comprises an enhancer.
24 . The polynucleotide of claim 22 , wherein the second regulatory element comprises a promoter and optionally comprises an enhancer.
25 . The polynucleotide of claim 21 , wherein the first regulatory element comprises an early promoter.
26 . The polynucleotide of claim 22 , wherein the second regulatory element is a late promoter.
27 . The polynucleotide of claim 22 , wherein the second regulatory element comprises an inducible control element, optionally the tet system, or a repressible control element, optionally the tetr system.
28 . A vector(s) comprising the polynucleotide(s) of claim 20 .
29 . The vector(s) of claim 28 , comprising one or more plasmid or viral vector.
30 . A cell or cell line or non-human animal, or progeny thereof, modified so as to contain the composition of claim 1 or a polynucleotide of claim 20 or a vector(s) of claim 28 .
31 . The non-human animal of claim 30 , which constitutively expresses the CRISPR Cas9-DD fusion.
32 . The non-human animal of claim 31 , wherein the non-human animal is a mouse.
33 . Progeny of the cell or cell line or non-human animal of claim 30 .
34 . A DD-CRISPR-Cas System comprising a composition of claim 1 , a polynucleotide of claim 20 , or a vector of claim 28 .
35 . A method of controlled targeting of a polynucleotide of interest in a cell comprising a DD-CRISPR-Cas9 system of claim 34 present in the cell whereby a guide polynucleotide of the DD-CRISPR Cas9 system targets the polynucleotide of interest; and having, in a controlled manner, a stabilizing ligand as to the DD present in the cell.
36 . A method of treatment of a subject in need thereof comprising administering a DD-CRISPR-Cas9 system of claim 34 to the subject whereby it is present in cell(s) of the subject whereby a guide polynucleotide of the DD-CRISPR Cas system targets a polynucleotide of interest involved in a condition of the subject, the targeting of which results in a treatment therefor; and having, in a controlled manner, a stabilizing ligand as to the DD present in cell(s) of the subject.
37 . A CRISPR-Cas9 complex comprising the composition of claim 1 , a guide polynucleotide and a nucleic acid target.