The PacBio-based long-read protocol was fully implemented and more than 50 evolved libraries have been sequenced at a depth of at least 20.000 individual clones. This data allowed us to e.g. identify when specific mutations emerged in the library and how quickly they were selected for. We also observed that 157 positions in the 324 amino acid long recombinase coding sequence had been selected to be altered during at least one evolved enzyme libraries. More interestingly, certain positions were found to change to different amino acids, depending on the selected target site the recombinases were evolved to recombine. In some extreme cases, eight different amino acids were found to be selected from the different projects. Hence, these “hotspots” are likely important specificity-determining residues. Overall, we have obtained excellent insight into the evolution dynamics of evolved recombinases. Using this data, we could further optimize the SLiDE procedure building first models to improve the platform. As a proof of concept for dual SSRs, we have evolved two recombinases that together excise a DNA fragment from the human genome. A manuscript describing the methods used to generate these enzymes and their molecular characterization has been published (PMID: 31745551). We have also identified an excellent target sequence for a dual SSR in the inverted repeats of the F8 gene and have generated individual recombinases that recombine their symmetric half-sites. Clones that together can recombine the final F8 target site have been identified. Efficiency and specificity tests of these candidate recombinases were performed in E.coli. Over 1000 individual recombinases were sequenced and their recombination efficiency quantified. The best 10 recombinases were further examined and tested at different recombinase induction levels. To investigate target site specificity, loxF8 variant sites were tested in the SLiDE vector system. Furthermore, the individual enzymes were tested on the asymmetric as well as on the symmetric target site. Moreover, the best candidate recombinase was expressed in bacteria carrying a human BAC clone covering the int1h region. Using a PCR assay we could confirm that upon expression of the recombinase, inversion at the expected position on the BAC took place. Hence, some excellent candidates could be identified that are now characterized further. The principle of designer-recombinase RMCE was demonstrated with loxLTR and loxBTR-specific recombinases. We have also identified suitable target sites flanking exon 8 of the F9 gene and have evolved SSRs that recombine these sites. Interestingly, we were able to identify single recombinase clones that can fulfill the full RMCE reaction, although the target half-sites are quite different from one another. Importantly, we could show that this recombinase can carry out the cassette exchange reaction in mammalian cells, when the cells were co-transfected with a donor and a receptor plasmid, plus a recombinase expression plasmid. Future work will focus on testing if this cassette exchange reaction can also be observed when the acceptor DNA is placed into the chromatin of the human genome. The pSLiDE-4R vector was built and shown to allow inducible expression of 4 different recombinases. We did not manage to make much progress on the RNA-guided SSR system, as we did not see enrichment of clones that only excised the SLiDE-vector upon expression of the gRNA. As an alternative, we are currently testing zinc-finger-SSR fusions. To place the zinc-fingers in frame within the recombinase coding sequence without disrupting recombinase function, we analyzed the structure of Cre and identified positions where the zinc-fingers could be inserted. Indeed, several of these Cre-based recombinases showed recombination activity on loxP. Unfortunately, when we inserted the zinc-finger motif into recombinases that had been evolved in our laboratory (e.g. Tre, Brec1), these enzymes showed no recombination activity on their respective target sites. To identify regions in these recombinases where insertions do not disrupt function, we are currently carrying out pentapeptide scanning mutagenesis for several evolved designer-recombinases.