Extended Data Fig. 6: D7-ZF off-target analysis.
From: Activation of recombinases at specific DNA loci by zinc-finger domain insertions
a Plasmid-based activity assay of D7-ZF on loxF8 and its extended versions. The extended sequences for the flanking genomic sequence for the ZFL from the left site (loxF8-flank-L) or the flanking genomic sequence for the ZFR from the right site (loxF8-flank-R) are indicated and color-coded. Activity of the wild-type D7 recombinase heterodimer is shown as a control. The test was performed at high induction level (100 ug/ml L-arabinose). The assay was performed three times (n = 3 biologically independent replicates). The upper band represents the unrecombined plasmid (line with two triangles), and the lower band represents the recombined plasmid (line with one triangle); M = Marker. b Schematic depiction of the bioinformatic loxF8-ZF off-target search. The previously predicted loxF8-like target sites8 were screened for the presence of both or one of the two potential ZFD DNA binding sites (position weight matrices of the predicted sites are shown on the left side for the ZFL and on the right side for the ZFR). c Unbiased experimental identification of putative human off-target sites by ChIP-Seq. ChIP-seq pileups at the loci of the selected peaks for the D7-ZF sample and GFP control are shown. Chromosomal positions and a size bar are indicated for each plot. d qPCR-based validation of putative D7-ZF binding sites identified by ChIP-seq. The GFP sample was used as a control. The heatmap displays the enrichment as a difference of the Cq in the input sample and in the IP sample for each tested peak. e Plasmid-based bacterial D7-ZF activity assay on the ten identified D7-ZF binding sites. M = Marker. The assay was performed three times (n = 3 biologically independent replicates).
