Method of Modifying Target Region in Host DNA and Selectable Marker Casette

A method of deleting a target region for deletion in a host DNA using a donor DNA:

• wherein the donor DNA comprising regions homologous to a 5′-side region outside of the target region for deletion in the host DNA, a 3′-side region outside of the target region for deletion in the host DNA and a first homologous recombination region inside of the target region for deletion in the host DNA, respectively, in this order, and further comprising a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region;
• 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 two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is deleted based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter.

A method of replacing a target region for replacement in a host DNA using a donor DNA:

• wherein the donor DNA comprising regions homologous to a 5′-side region outside of the target region for replacement in the host DNA, a 3′-side region outside of the target region for replacement in the host DNA and a first homologous recombination region inside of the target region for replacement in the host DNA, respectively, in this order, comprising a desired DNA sequence between the region homologous to the 5′-side region and the region homologous to the 3′-side region, and further comprising a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region;
• 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 two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is replaced with the desired DNA sequence based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter.