Figure 1.

Detection of ZFN-induced mutations at a GFP transgene in soybean. A, The position of the OPEN ZFN target site is represented by a gray rectangle. The target sequence of both left and right ZFAs recognize a 9-bp sequence (indicated in bold). A strategy involving the restriction enzyme BccI and a PCR assay was used to enrich and identify mutated sequences. B, Amplicons from the PCR assay were cloned into pGem T-easy and subsequently amplified by colony PCR using the GFP-specific primers (Supplemental Table S3). The sequencing of PCR products revealed large deletions ranging from 27 to 71 bp. The enrichment of mutated GFP sequence was biased toward large deletions at the 5′ region of the target site since the BccI recognition site CCATC was located on the left ZFA recognition sequence. Typically the restriction site is situated in the middle of the target site, as the majority of obtained indels are minor (1–10 bp) and occur in the spacer region.