Rapid Mutation by OPEN ZFNs Figure 2. Toxicity and teratogenicity of OPEN and B1H-selected ZFNs in zebrafish embryos. Percentages of dead, deformed (‘‘monster’’), and normal embryos following injection with the amounts of ZFN RNAs indicated are shown. Percentages were calculated from the number of embryos (n) indicated. doi:10.1371/journal.pone.0004348.g002 org/Danio_rerio/Info/Index). In total, we found 315,186 poten- tial ZFN sites in these 29,291 gene transcripts (Supplemental Tables S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25). Importantly, we identified one or more potential OPEN ZFN sites (mean of ,4.5 sites) in the first three coding exons of 25,174 of these gene transcripts and two or more potential OPEN ZFN sites (mean of ,5.4 sites) in the first three-coding exons of 20,418 gene transcripts (Table 3). These results suggest that OPEN could potentially be used to generate ZFNs for as many as 86% of the transcripts encoded in the zebrafish genome. Discussion In this report, we used the recently described OPEN zinc finger engineering method to rapidly generate ZFNs that can modify endogenous zebrafish gene targets with high efficiency. In less than two months time, we generated ZFNs for target sites in five different biologically important genes. The five pairs of ZFNs we tested can efficiently generate mutations at their intended endogenous gene target in somatic zebrafish cells. In addition, we demonstrated that mutations induced by OPEN ZFNs can be efficiently transmitted through the germline. These results suggest that only a small number of fish need to be screened to identify founders, consistent with previously published results [27,28]. Our results nearly triple the total published number of endogenous zebrafish genes successfully modified using ZFNs from three (kdr, golden, and ntl) to eight and demonstrate that OPEN is a highly effective ZFN engineering method for creating targeted mutations in zebrafish. In silico analysis indicates that as many as 25,174 zebrafish gene transcripts have one or more potential OPEN ZFN target sites in their first three coding exons and as many as 20,418 transcripts have two or more sites in the first three coding exons. Our PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4348