Recombinase made to measure to recombine asymmetric target sites in a plurality of retrovirus strains

摘要

Method for preparing an expression vector encoding a custom-made recombinase, said custom-made recombinase being able to recombine asymmetric target sequences within the LTR of DNA from a plurality of retrovirus strains that are strains of HIV-1, comprising the following steps: (a) identify sequences with a homology of at least 30% with respect to the left hemisphere sequence and right hemisphere sequence of at least one recombinase target site known in the LTR sequence of the proviral DNA of a plurality of retrovirus strains, in which the homologous sequences are separated by a spacer of 5 to 12 nucleotides and in which the asymmetric target sequence is found in a plurality of retrovirus strains, in which the asymmetric target sequence presents the sequence set forth as SEQ ID No. 1, (b) identifying two sequences, in which the first sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the left hemisphere of said known target site and referred to as "hemisphere sequence 1" and in which the second sequence corresponds to the sequence of the asymmetric target sequence from step (a) homologous to the right hemisphere and referred to as "hemisphere sequence 2"; (c) determining the nucleotides within the sequences of stage (b) that differ from the corresponding homologous left hemisphere and right hemisphere sequences of said at least one known homologous target site of stage (a); (d) generating a first subset of two target nucleic acids comprising target sequences, in which the first target sequence is called subsite 1 and comprises, adjacent to each other and in order 5 'to 3', the hemisphere sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of hemisphere sequence 1, and in which the second target sequence is called subsite 2 and comprises, adjacent to each other and in order 5 'to 3 ', an inverted repeat of the hemisphere sequence 2, the spacer sequence of the asymmetric target sequence and the hemisphere sequence 2 of step 30 (b), (e) generate a second subset of target nucleic acids comprising target sequences modified on the basis of the target sequences of the first subset of step (d), in which, in the sequences based on subsite 1, in the left hemisphere sequence, a part of the nucleotides that differ from the corresponding homologous hemisphere sequence of at least one known target site of step (a) is replaced by the natural nucleotides found in said known target site, until said hemisphere sequence contains one, two or three nucleotides that differ from said known target site, in which the right hemisphere of said modified target sequence is formed by an inverted repetition of said modified left hemisphere sequence, which is separated from said modified left hemisphere sequence by the spacer sequence of the asymmetric target sequence, and wherein, in the sequences based on subsite 2, in the right hemisphere sequence, a part of the nucleotides that differ from the corresponding homologous hemisphere sequence of said at least one known target site of step (a ) is replaced by the natural nucleotides found in said known target site, until said sequence of h emisitio contains one, two or three nucleotides that differ from said known target site, in which the left hemisphere of said modified target sequence is formed by an inverted repetition of said modified right hemisphere sequence, which is separated from said modified sequence of right hemisphere by the spacer sequence of the asymmetric target sequence, so that in all the modified hemisphere sequences originated from a target sequence of the first subset of step (d) taken together, all the differentiated nucleotides can be found , while none of said modified hemisphere sequences alone comprises all the differentiated nucleotides, (f) separately applying the molecular evolution directed to at least one recombinase that recognizes a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as substrate, (g) mixing the collections of recombinases evolved in step (f), in which all collections of recombinases evolved on sequences based on subsite 1 are combined and mixed, and in which all collections of recombinases evolved on sequences based on subsite 2, they are combined and mixed; (h) apply the molecular evolution directed to the mixed collections obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate; (i) mix the collections of recombinases evolved in step (h); (j) applying the substrate bound protein evolution of the mixed collection obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase that is active on the asymmetric target sequence within the retroviral DNA LTR of step (a); (k) isolating the nucleic acid encoding said at least one recombinase obtained in step (j) from the collection; and (l) cloning the nucleic acid obtained in step (k) into a suitable expression vector.