Patent ID: 8852943

Claim:
A method of deleting a target region in a host DNA using a donor DNA: wherein the donor DNA comprises regions homologous to (a) a 5′-side region outside of the target region in the host DNA, (b) a 3′-side region outside of the target region in the host DNA and (c) a first homologous recombination region inside of the target region in the host DNA, wherein (a), (b) and (c) are, respectively, in that order, and, between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region further comprises a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene, wherein the second selectable marker gene is expressed under the control of the expression-inducing promoter; which method comprises the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5′-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, inducing expression of said second selectable marker gene, and selecting a host whose target region is deleted based on a lack of expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter, wherein said second selectable marker gene does not remain in said host DNA after said second step of performing homologous recombination.