‘‘donor template’’ (an approach known as ‘‘gene targeting’’) can be used to introduce a specific mutation near the break or to insert a DNA sequence at the the break. Recent proof-of-principle studies have shown that ZFNs can also be used to create targeted NHEJ-mediated knockout mutations in endogenous zebrafish genes. Wolfe and Lawson created ZFN-induced knockouts in the kdr gene [27] while Amacher and colleagues mutated the golden and ntl genes [28]. These results demonstrate that ZFNs can provide an important genetic capability previously unavailable to researchers in the zebrafish field and have created much excitement in the community. An important question raised by these groundbreaking studies is how can the typical zebrafish researcher generate the customized ZFNs required to practice this targeted knockout technology [29]. The Wolfe and Lawson kdr ZFNs [27] were made using a modified version of a previously described two- stage optimization strategy [30]. This approach is very difficult for the non-specialist scientist to practice because it requires the construction and interrogation of three partially randomized zinc finger libraries and of a secondary recombinant library derived from the outputs of the initial three libraries. The ntl and golden ZFNs used by Amacher and colleagues were constructed using a proprietary engineering platform developed by Sangamo BioSciences, Inc. [28]. ZFNs made by this proprietary method can be purchased from Sigma-Aldrich but the high fee charged per ZFN pair [31] may make it difficult for most labs to purchase ZFNs for more than one or two genes of interest. A third method previously used to make ZFNs (for use in other cell types) is the ‘‘modular assembly’’ approach in which zinc fingers with pre-selected specificities are joined together [32–35]. However, a recent large-scale assessment of the modular assembly method demonstrated that it is highly inefficient with a success rate for making functional ZFN pairs that is at best ,6% [36]. The Zinc Finger Consortium recently described the develop- ment and validation of a rapid, highly effective, and publicly available method for engineering zinc finger arrays termed OPEN (for Oligomerized Pool ENgineering) [26]. OPEN requires the construction of only a single recombinant zinc finger library (smaller than 106 in size) and yields ZFNs that function with high efficiencies in human and plant cells [26]. The method accounts for the context-dependent DNA-binding activities of zinc fingers, a parameter that previous studies have suggested is important for creating arrays with high DNA-binding affinities and specificities [30,37–42]. In direct comparisons, OPEN exhibited a much higher success rate for yielding functional ZFNs than the modular assembly method [26]. In indirect comparisons performed with different target sites, ZFNs made by OPEN also exhibited activities and toxicities comparable to ZFNs made by the proprietary Sangamo BioSciences approach [26]. In this study, we use a modified and more rapid version of OPEN to generate ZFNs for five endogenous zebrafish gene targets. We show that these OPEN ZFNs efficiently induce indel mutations in their respective endogenous gene targets in somatic zebrafish cells. In addition, we demonstrate germline transmission of ZFN-induced mutations for four of the five gene targets. Finally, we use in silico analysis to show that one or more potential OPEN ZFN target sites can be found within the first three coding exons of more than 25,000 transcripts derived from endogenous zebrafish genes. Our results demonstrate that OPEN can rapidly generate ZFNs for efficient mutation of endogenous genes in zebrafish and provide strong additional support for its use with this important model organism. Rapid Mutation by OPEN ZFNs Results Using OPEN to engineer zinc finger arrays for endogenous zebrafish gene targets We used the OPEN method to engineer zinc finger arrays for potential ZFN target sites in five different endogenous zebrafish genes: dopamine transporter (dat), hypoxia-inducible factor 1a (hif1aa), telomerase, transferrin receptor 2 (tfr2), and gridlock. The targeted genes differ widely in size, genomic location, and functional class (channel, receptor, enzyme, transcription factor) and were selected for their relevance to ongoing zebrafish research projects or for their general utility for the zebrafish community. We used the web-based ZiFiT v3.0 software program (http://bindr.gdcb.iastate.edu/ZiFiT/) [26] to identify potential target sites in the coding sequences of these genes. Ten selections (one for each half-site in the five full ZFN target sites) were performed using an improved, more rapid version of our recently described OPEN method (Figure 1). Alterations made to the original method included miniaturization of the selections so that they can be performed using multi-channel pipets, multi-well (24- well) blocks, and smaller amounts of solid and liquid media (see Materials and Methods). These alterations have led to a substantial increase in the speed of the procedure: as many as 48 selections can now be completed by two individuals in less than 8 weeks time. The OPEN selections we performed successfully yielded multiple active zinc finger arrays for all 10 target half-sites (Table 1). As before, we defined successful arrays as those that can activate transcription of a lacZ reporter gene by three-fold or more in the B2H system [26]. Previous studies have shown that zinc finger arrays which activate transcription above this threshold in the B2H system possess high affinity and high specificity for their cognate DNA binding site [30]. For comparison, we also tested the zinc finger arrays from the kdr ZFNs previously made by Wolfe and Lawson [27] on their respective target half-sites. Interestingly, we found that although one of the arrays (ZFP1) activated transcription more than three-fold in the B2H system, the other (ZFP2) failed to show any activation (Table 1). The lower activity of the kdr ZFP2 zinc finger array in the B2H system may be due to low DNA-binding specificity as well as to the lower stringency of the bacterial one-hybrid (B1H) system used to select the kdr- targeted zinc finger arrays (see Discussion below). Efficient somatic cell mutation of endogenous zebrafish genes using OPEN ZFNs We next tested the abilities of zinc finger arrays obtained by OPEN to induce mutations when expressed as ZFNs in somatic zebrafish cells. To do this, we chose one zinc finger array for each ZFN target half-site and tested pairs as ZFNs (highlighted in bold italics in Table 1). To test the robustness of our OPEN selections, we chose zinc finger arrays with high (but not always the highest) B2H fold-activation for testing as ZFN pairs; however, all arrays tested met the minimum three-fold B2H activation threshold described above. DNA fragments encoding these zinc finger arrays were cloned into ZFN expression vectors previously constructed by the Joung lab (see Materials and Methods for details) [26]. The ten resulting vectors encode ZFNs consisting of a FLAG epitope tag, an SV40 nuclear localization signal, and a zinc finger array fused to an obligate heterodimeric FokI nuclease domain [43]. These vectors also harbor a bacteriophage T7 promoter positioned upstream of the ZFN coding sequence. As a positive control, we also constructed two additional ZFN expression plasmids which encoded obligate heterodimeric ZFNs harboring the Wolfe/ Lawson kdr zinc finger arrays (ZFP1 and ZFP2) [27]. We note that these control kdr ZFN plasmids are identical to our OPEN ZFN expression vectors except for the sequences encoding the zinc PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4348