The present invention relates to a method of producing a eukaryotic cell carrying a modified target sequence in its genome, the method comprising the step: (a) introducing into the cell a fusion protein comprising a DNA-binding domain of a Ralstonia solanacearum TALE-like protein (RTL) effector protein and a non-specific cleavage domain of a restriction nuclease or a nucleic acid molecule encoding the fusion protein in expressible form, wherein the fusion protein specifically binds within the target sequence and introduces a double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the modification of the target sequence is by homologous recombination with a donor nucleic acid sequence further comprising the step: (b) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal or vertebrate carrying a modified target sequence in its genome. Furthermore, the present invention relates to a fusion protein comprising a RTL effector protein and a non-specific cleavage domain of a restriction nuclease (RALEN).
