Conditioned dna modifying enzyme comprising heterologous dna binding domain

Method of evolving a DBD on a target sequence of interest, comprising the steps of:

• (i) creating a library of variants of the DBD;
• (ii) cloning the library of step (i) into expression vectors comprising a first region encoding a DNA recombining enzyme, a second region comprising a first target site of said DNA recombining enzyme and regions flanking said first target site, and a third region comprising a second target site of said DNA recombining enzyme and regions flanking said second target site, such that a DBD is inserted directly or via peptide linkers into the DNA recombining enzyme, wherein the first, second and third regions are separated from another;
• (iii) introducing the expression vectors into host cells and culturing the host cells, thereby expressing the encoded DNA recombining enzyme comprising the DBD;
• (iv) isolating plasmids from the cell culture of step (iii) and determining whether the DNA recombining enzyme catalyzed a recombination reaction at both target sites on the vector;
• (v) amplifying the DBD of those plasmids that were found to encode a DNA recombining enzyme comprising the DBD showing recombination activity using error-prone PCR to generate a new library of variants of the DBD;
• (vi) repeating steps (ii) to (iv) with the library of step (v).

A DNA modifying enzyme comprising an insertion of a heterologous DNA Binding Domain (DBD), wherein:

• (i) the DNA modifying enzyme is Cre or a Cre-derived recombinase and the DBD is inserted between amino acid positions 278 and 279;
• or
• (ii) the DNA modifying enzyme is Vika or a Vika-derived recombinase and the DBD is inserted between amino acid positions 172 and 173;
• or
• (iii) the DNA modifying enzyme comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:
• 5, 9 to 13, 19, 20, 22, 30, 41, 42, 51, 64 and 91 to 200.