Fig. 3

Improved second generation of ZFN parasites and genotyping of resultant SI populations. a Design of Sp2ZFN and Ls2ZFN. zfnLcm (blue) is a codon-modified version of zfnL to maximise codon difference between zfnL and zfnR (grey). Sp2ZFN and Ls2ZFN drive expression of both ZFNs fused by the 2A skip peptide (red) from a single promoter. The mgfp (green) is a codon-modified version of egfp that carries a silent mutation within the most frequently observed microhomology in SpZFN SI (Fig. 2c). b PCR amplicons of ZFN loci and the mgfp gene are shown for Sp2ZFN c1 as well as Ls2ZFN c1 and LsZFN SI 1–9. Expected sizes are 3091 bp and 3479 bp for ZFN loci in Sp2ZFN and Ls2ZFN, respectively. Expected size of mgfp is 837 bp. Only Ls2ZFN SI 3 showed a slightly smaller size for mgfp. c Alignment of all mgfp genes. Only Ls2ZFN SI 3 showed a deletion of 81 bp. Binding sites of ZFNs are shown in colour if present. The microhomology region of 4 bp implicated in repair is highlighted in colour and with a red background. d Overview of the genomic loci found in all Ls2ZFN SI parasites. Only Ls2ZFN SI 3 is changed in respect to the original clone Ls2ZFN c1. No SI parasites were observed for Sp2ZFN