Figure 1: Disruption of an eGFP transgene in G. bimaculatus using designed ZFNs.

(a) Illustration of the binding and function of ZFNs and TALENs. Following the introduction of double-stranded breaks (DSB) in the DNA between targeted binding sites, these breaks are either repaired and digested again, or NHEJ can occur and deletions or insertions can result. In the latter case, gene function of the targeted gene is disrupted, and subsequent binding of ZFNs or TALENs does not result in FokI dimerization or the generation of DSBs. (b) Structure of the pBGact-eGFP transgene that is under the transcriptional control of the Gryllus cytoplasmic actin promoter (Gact) and flanked by 5′ and 3′ inverted terminal repeats (ITRs). The 9-bp binding sites for the ZFNs, eGFP-R and eGFP-L are in the boxes shown. (c) Detection of induced mutations into genomic DNA cleaved by Surveyor nuclease that was collected from embryos injected with both eGFP-R and eGFP-L mRNAs or eGFP-R mRNA alone (control) at 7 days postinjection. A product cleaved by Surveyor nuclease was detected in the genomic DNA from embryos injected with eGFP-R/L mRNAs (arrowhead). (d) Bright field (left panel) and fluorescence (right panel) phenotypes of G₁ crickets injected with eGFP ZFN mRNA at 2 days after egg laying. While the embryos appeared to develop normally, some embryos did not exhibit eGFP fluorescence (indicated with arrows). Scale bar, 2.5 mm. (e) Sequence analysis of eGFP mutant alleles induced by eGFP ZFN mRNAs. Wild-type (WT) sequences are shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Asterisks indicate knockout mutations that included frame-shift and stop codon insertions (underlined).