Fig. 4: Off-target analysis of F8 recombinases.

a Bacterial plasmid-based activity of the D7 heterodimer on predicted asymmetric and symmetric loxF8-like sites. The upper band represents non-recombined plasmid DNA (line with two triangles) whereas the lower band shows the recombined form of the plasmid (line with one triangle). The same applies for other gel pictures shown in this figure. b Schematic overview of physical linking of recombinases and the predicted activity on asymmetric and symmetric target sites. c Overview of the linker selection using alternating SLiDE. First, clones are selected for activity on loxF8. In the next two selection rounds these recombinases are selected against activity on symmetric target sites. Alternating the target sites and the selection of the linked recombinases allows for selection of clones with desired activity profiles (activity on loxF8, no activity on loxF8-L, and loxF8-R). d Bacterial plasmid-based activity of the linked RecF8 dimer on predicted human asymmetric and symmetric loxF8-like sites. e qPCR-based quantification of the genomic inversion efficiencies at the loxF8 locus after treatment with indicated recombinases in HEK293T cells (n = 3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). n.s. = not significant, unpaired two-sided t-test (two-stage step up method, Prism 8). f qPCR-based validation of putative binding sites identified by ChIP-seq. The RecF8 sample is compared to the GFP control sample. Enrichment is displayed by a heatmap of the difference of the Cq in the input sample and in the IP sample for each peak. g Bacterial plasmid-based RecF8 activity assay of the twelve RecF8-binding sites and on oxF8. The marker lane (M) for 4 kb and 5 kb is shown with arrows. h Venn diagrams showing counts of structural variants detected after whole-genome sequencing of recombinase-treated patient iPSCs, as well as in non-treated cells, in comparison to the reference genome. The diagram on the left represents counts of all called variants. The right diagram shows counts of deletions, translocations and inversions that are potentially caused by indicated recombinase activity. Source data are provided as a Source data file.