Peptide-mediated delivery of RNA-guided endonuclease into cells
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.
1. A method for modifying a target site in the genome of a microbial cell, the method comprising providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to the microbial 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, wherein the guide polynucleotide and CPP-Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double-strand break at the target site in the genome of the microbial cell.
2. The method of claim 1 , wherein the modifying at said target site includes (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).
3. The method of claim 1 , further comprising identifying at least one microbial cell that has a modification at the target site, wherein the modification at the target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).
4. The method of claim 1 , 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.
5. The method of claim 1 wherein said Cas endonuclease and CPP are covalently linked to each other.
6. The method of claim 1 , wherein said Cas endonuclease and CPP are non-covalently linked to each other.
7. The method of claim 1 , wherein the guide polynucleotide comprises
(i) a first nucleotide sequence domain that is complementary to a nucleotide sequence in said target site, and
(ii) a second nucleotide sequence domain that interacts with a Cas endonuclease,
wherein the first nucleotide sequence domain and the second nucleotide sequence domain are composed of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or a combination thereof.
8. The method of any preceding claims, wherein the guide polynucleotide is composed of ribonucleic acids (RNA).
9. The method of claim 1 , wherein the guide polynucleotide/Cas endonuclease-CPP complex is a RNA-guided endonuclease (RGEN)-CPP complex.
10. The method of claim 9 , wherein the RGEN comprises a CRISPR-associated (Cas) protein-9 (Cas9) amino acid sequence.