Rapid Mutation by OPEN ZFNs Figure 1. The OPEN Zinc Finger Selection Method. (A) Schematic overview of OPEN selection for a target DNA site. Zinc finger domains are shown as spheres and associated 3 bp subsites as rectangles. Details provided in the text and in Maeder et al., Mol. Cell 2008. (B) Schematic of the bacterial two-hybrid (B2H) system. ZFP = zinc-finger protein. X and Y = arbitrary interacting proteins. doi:10.1371/journal.pone.0004348.g001 finger arrays. We transcribed RNA from each of these 12 ZFN expression plasmids and performed poly A-tailing of the RNA as described in Materials and Methods. In an initial control experiment to test whether our ZFN vectors and experimental conditions would work efficiently in zebrafish, we injected ,100 embryos each with 100 pg of purified RNA made from the pair of vectors encoding the Wolfe/Lawson kdr ZFNs (50 pg of RNA encoding each ZFN). As shown in Figure 2, we observed that approximately 79% of the embryos were dead or exhibited a highly deformed ‘‘monster’’ phenotype, consistent with previously published experiments performed with these ZFNs [27]. Furthermore, we observed that we could not inject more than 100 pg of RNA/embryo without causing death in almost all embryos (data not shown). To assess whether targeted mutagenesis of kdr occurred at the somatic cell level, we harvested genomic DNA from a pool of 10 embryos two days post-injection and sequenced the region of the kdr gene targeted by the Wolfe/ Lawson ZFNs using a limited cycle PCR/DNA-sequencing method previously described and validated by the Joung lab for quantitation of mutations in a population of alleles [26]. As shown in Figure 3A, 10% of the kdr alleles we sequenced harbored insertions or deletions at the site of the ZFN-induced DSB, a PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4348