Crispr having or associated with destabilization domains

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.

What is claimed: 
     
         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.