Fig. 4

ZFN parasites with optimised expression timing and genotyping of SI populations. a Design of TrapZFN and Uis4ZFN. Both parasite lines express ZFNs fused with a 2A skip peptide under control of the respective promoters of trap and uis4. b PCR products of the zfn loci and the mgfp gene are shown for parental clones Uis4ZFN c1 and Uis4ZFN SI 1–3 and TrapZFN c1 as well as TrapZFN SI 1–2. The expected size of the zfn genes was 3303 bp for Uis4ZFN, 3321 bp for TrapZFN and 837 bp for mgfp. The ZFN loci were all identical in size, whereas Uis4ZFN SI 1–3 and TrapZFN SI 1–2 showed shorter PCR products for mgfp of varying sizes. c Alignment of all mgfp genes. TrapZFN SI 1 had a deletion of 369 bp; in TrapZFN SI 2 474 bp were deleted. Binding sites of ZFNs are coloured and the microhomology regions implicated in repair of 6 and 7 bp, respectively, are highlighted in colour and with a red background. Both Uis4ZFN SI 1–2 and SI 3 had a deletion of the same 81 bp. Microhomology regions implicated in repair are 10 bp, including one mismatch highlighted in white for SI 1–2 and 7 bp for SI 3. d Overview of the genomic loci found in both TrapZFN SI 1–2 and Uis4ZFN SI 1–3. Both TrapZFN SI parasites have big deletions in the mgfp gene with respect to the original clone TrapZFN c1; Uis4ZFN SI parasites show a small deletion in the mgfp gene