CRISPR-Cas9-inhibiting polypeptide
View Patent ↗CRISPR-Cas9-inhibiting polypeptides, cells expressing the polypeptides, and polynucleotides encoding the polypeptides are provided. The Cas9-inhibiting polypeptides can be used in many aspects to inhibit unwanted Cas9 activity. For example, one or more Cas9-inhibiting polypeptides can be used to regulate Cas9 in genome editing, thereby allowing for some Cas9 activity prior to introduction of the Cas9-inhibiting polypeptide.
1 . A method of inhibiting a Cas9 polypeptide in a cell, the method comprising, introducing a Cas9-inhibiting polypeptide into a cell, wherein:
the Cas9-inhibiting polypeptide is heterologous to the cell,
the Cas9-inhibiting polypeptide is at least 95% identical to SEQ ID NO:1; and
the Cas9 polypeptide is present in the cell before the introducing or the Cas9 polypeptide is introduced into the cell after the introducing,
thereby inhibiting the Cas9 polypeptide in a cell.
2 . The method of claim 1 , comprising contacting the Cas9 inhibiting polypeptide with a Cas9 polypeptide in the cell.
3 . The method of claim 1 , wherein the Cas9-inhibiting polypeptide comprises SEQ ID NO:1.
4 . The method of claim 1 , wherein the cell comprises the Cas9 polypeptide before the introducing.
5 . The method of claim 4 , wherein the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding the Cas9 polypeptide.
6 . The method of claim 5 , wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell prior to the introducing.
7 . The method of claim 1 , wherein the cell comprises the Cas9 polypeptide after the introducing.
8 . The method of claim 7 , wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell after to the introducing.
9 . The method of claim 1 , wherein the introducing comprises expressing the Cas9-inhibiting polypeptide in the cell from an expression cassette that is present in the cell and heterologous to the cell, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide.
10 . The method of claim 9 , wherein the promoter is an inducible promoter and the introducing comprises contacting the cell with an agent that induces expression of the Cas9-inhibiting polypeptide.
11 . The method of claim 1 , wherein the introducing comprises introducing an RNA encoding the Cas9-inhibiting polypeptide into the cell and expressing the Cas9-inhibiting polypeptide in the cell from the RNA.
12 . The method of claim 1 , wherein the introducing comprises inserting the Cas9-inhibiting polypeptide into the cell or contacting the cell with the Cas9-inhibiting polypeptide.
13 . The method of claim 1 , wherein the method occurs ex vivo.
14 . The method of claim 13 , wherein the cells are introduced into a mammal after the introducing and contacting.
15 . A cell comprising a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is heterologous to the cell and the Cas9-inhibiting polypeptide is at least 95% identical to SEQ ID NO:1.
16 . A polynucleotide comprising a promoter operably linked to a nucleic acid encoding a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide has at least 95% identity to SEQ ID NO: 1, and wherein the promoter is heterologous to the nucleic acid.
17 . A vector comprising the polynucleotide of claim 16 .
18 . A pharmaceutical composition comprising the polynucleotide of claim 16 .
19 . A delivery vehicle comprising the polynucleotide of claim 16 .
20 . The delivery vehicle of claim 19 , wherein the delivery vehicle is a liposome or nanoparticle.