Fig. 1

DEQSeq identifies favorable designer-recombinases. a Workflow overview. Evolved recombinases are cloned together with a unique molecular identifier (UMI) into a vector containing loxF8 target sites. E. coli are transformed with the barcoded plasmids. The number of transformed bacteria is determined by counting colonies on agar plates with an antibiotic matching the resistance gene on the plasmids. Calculated from the transformation efficiency, a defined number of transformed bacteria is cultured. Recombinase expression is induced by the addition of L-arabinose, which may result in the recombination of the target sites on the plasmid. The plasmid DNA is isolated and the recombinase genes and their UMIs are subcloned into pEVOs with different target sites (HG1, HG2, and HG2L) and cultured as before. From all the cultured plasmids, the region containing the gene, the UMI, and the target sites are excised and sequenced with nanopore sequencing. The sequences of the UMIs are computationally clustered based on similarity, resulting in groups of sequences corresponding to the different variants. From these groups, the recombination events on the different target sites are counted and accurate recombinase sequences are generated. b DEQSeq screen of loxF8 recombinases on the four target sites as indicated (loxF8, HG1, HG2, HG2L). UMI-clusters containing evolved recombinases are indicated as blue points and D7 control clusters are indicated in black. Marked with numbers are recombinase variants that were further evaluated. Enzyme expression was induced with 1 µg/ml L-arabinose on loxF8 and 100 µg/ml L-arabinose on all other target sites