Peptide-mediated delivery of rna-guided endonuclease into cells

Abstract

A composition is disclosed that comprises at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. The RGEN protein component of an RGEN protein-CPP complex in certain embodiments can be associated with a suitable RNA component to provide an RGEN capable of specific DNA targeting. Further disclosed are compositions comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one CPP, as well as methods of delivering RGEN proteins into microbial cells, as well as methods of targeting DNA with RGENs.

What is claimed is: 
     
         1 . A composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP),
 wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, and   wherein said RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell.   
     
     
         2 . The composition of  claim 1 , wherein the protein component of the RGEN is associated with at least one RNA component that comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. 
     
     
         3 . The composition of  claim 2 , wherein the RNA component comprises a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) operably linked to a trans-activating CRISPR RNA (tracrRNA). 
     
     
         4 . The composition of  claim 2 , wherein the RGEN can cleave one or both DNA strands at the target site sequence. 
     
     
         5 . The composition of  claim 1 , wherein the RGEN comprises a CRISPR-associated (Cas) protein-9 (Cas9) amino acid sequence. 
     
     
         6 . The composition of  claim 1 , wherein the RGEN protein component and CPP are covalently linked. 
     
     
         7 . The composition of  claim 1 , wherein the RGEN protein component and CPP are non-covalently linked. 
     
     
         8 . The composition of  claim 1 , wherein the CPP is cationic or amphipathic. 
     
     
         9 . The composition of  claim 1 , wherein the CPP comprises:
 (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein,   (ii) a CPP having 6 or more contiguous arginine residues,   (iii) a transportan-10 (TP10) CPP, or   (iv) a CPP from a vascular endothelium cadherin protein.   
     
     
         10 . The composition of  claim 1 , wherein said RGEN protein-CPP complex can traverse a cell wall and cell membrane of a microbial cell. 
     
     
         11 . A cell comprising the composition according to  claim 1 . 
     
     
         12 . A method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell, said method comprising:
 contacting the microbial cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP),   wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex,   wherein said RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the microbial cell.   
     
     
         13 . The method of  claim 12 , wherein:
 (i) the composition further comprises at least one RNA component that is associated with the protein component of the RGEN; or   (ii) the microbial cell comprises the RNA component, wherein the RNA component associates with the protein component of the RGEN after the RGEN protein-CPP complex enters the microbial cell;   wherein the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence.   
     
     
         14 . The method of  claim 13 , wherein the RGEN can cleave one or both DNA strands at the target site sequence. 
     
     
         15 . The method of  claim 14 , wherein the microbial cell further comprises a donor polynucleotide comprising at least one sequence homologous to a sequence at or near the target site sequence, and wherein the donor polynucleotide integrates at or near the target site sequence by homologous recombination. 
     
     
         16 . A polynucleotide sequence comprising a nucleotide sequence encoding an RGEN protein-CPP fusion protein that comprises a protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein optionally, said nucleotide sequence is operably linked to a promoter sequence. 
     
     
         17 . A method of producing an RGEN protein-CPP fusion protein comprising:
 (a) providing the polynucleotide sequence of  claim 16 ;   (b) expressing the RGEN protein-CPP fusion protein from the polynucleotide sequence, thereby producing the RGEN protein-CPP fusion protein, wherein said expressing is optionally performed in a cell; and   (c) optionally, isolating the RGEN protein-CPP fusion protein produced in step (b).   
     
     
         18 . A composition comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein said guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell. 
     
     
         19 . A method for modifying a target site in the genome of a cell, the method comprising providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to the cell, wherein said guide polynucleotide, Cas endonuclease and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein said guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of the microbial cell