Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains

A method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, comprising the steps of

• (a) identifying SEQ ID NO:1 sequences with a homology of at least 30% to the left half-site sequence and the right half-site sequence of at least one known recombinase target site in the sequence of the LTR of proviral DNA of a plurality of retrovirus strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and represent an asymmetric target sequence within the LTR of proviral DNA, and wherein the asymmetric target sequence is found in a plurality of retrovirus strains;
• (b) identifying two sequences, wherein the first sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the left half-site of said known target site and is referred to as “half-site sequence 1”, and wherein the second sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the right half-site and is referred to as “half-site sequence 2”;
• (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the at least one known homologous target site of step (a);
• (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b);
• (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d),
• wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and
• wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence,
• such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides,
• (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate;
• (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;
• (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate;
• (i) shuffling the recombinase libraries evolved in step (h);
• (j) applying molecular directed evolution on the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the proviral DNA of step (a);
• (k) isolating the nucleic acid encoding the least one recombinase obtained in step (j) from the library;
• and
• (l) optionally, cloning the nucleic acid obtained in step (k) into a suitable expression vector.