Genome editing with split Cas9 expressed from two vectors
The present invention relates to a method for regulating gene expression, comprising introducing into a cell each of a recombinant vector which expresses a first domain comprising N-terminus of a Cas9 protein, and a recombinant vector which expresses a second domain comprising C-terminus of a Cas9 protein, a composition comprising the recombinant vectors, a kit for regulating gene expression, and a method for intracellular production of Cas9 protein. Moreover, the present invention relates to a transformed cell introduced with a viral vector which packages the first domain, and a viral vector which packages the second domain, and to a composition comprising a virus produced therefrom.
1. A method for introducing a targeted mutation into a genome, comprising introducing into an isolated cell (i) a recombinant vector, which expresses a first domain comprising the N-terminus of a Cas9 protein, and (ii) a recombinant vector which expresses a second domain comprising the C-terminus of a Cas9 protein,
wherein the first domain is encoded by the nucleotide sequence of SEQ ID NO: 3, and the second domain is encoded by the nucleotide sequence of SEQ ID NO: 5,
wherein the first domain and the second domain are constructed by cleaving a middle portion of the sequence of SEQ ID NO.:10 in the Cas9 protein, in which SG amino acids and QG amino acids are respectively linked to the first domain and the second domain.
2. The method of claim 1 , wherein the Cas9 protein is derived from any one selected from the group consisting of Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, and Streptococcus mutans.
3. The method of claim 1 , wherein the recombinant vector is a plasmid vector, a cosmid vector, or a viral vector.
4. The method of claim 3 , wherein the viral vector is selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and a herpes simplex virus vector.
5. The method of claim 1 , further comprising fusing the first domain and the second domain, which are expressed from each of the introduced recombinant vectors, to form the Cas9 protein.
6. The method of claim 1 , wherein the first domain and the second domain each comprises an NLS (nuclear localization signal) sequence, an hemagglutinin (HA) tag sequence, a splicing donor sequence, a splicing acceptor sequence, or a combination thereof.
7. The method of claim 1 , wherein a sequence-specific guide RNA is introduced into the cell.
8. The method of claim 7 , wherein introducing each vector and the guide RNA is performed in a simultaneous or sequential manner.