Rapid Mutation by OPEN ZFNs Table 3. Summary of potential OPEN ZFN target sites identified in zebrafish transcripts Number of transcripts with: …in the first coding exon …in the first two coding exons …in the first three coding exons One or more potential ZFN sites… Two or more potential ZFN sites… 14,623 9,292 21,781 15,961 25,174 20,418 A total of 29,291 gene transcripts derived from the Ensembl release 51 Danio rerio database were analyzed for potential OPEN ZFN target sites (see Materials and Methods for additional details). doi:10.1371/journal.pone.0004348.t003 form can be used to engineer ZFNs for a high percentage of zebrafish genes. The reagents we used to construct and express ZFNs in our experiments are part of the OPEN Zinc Finger Consortium platform for engineering zinc finger arrays. Zinc finger arrays constructed by OPEN can be excised as XbaI/BamHI fragments and cloned directly into expression plasmids which then encode FLAG-tagged, NLS-tagged, obligate heterodimeric ZFNs. ZFN- encoding RNA can be directly transcribed from these plasmids using T7 RNA polymerase. OPEN Zinc Finger Consortium reagents are publicly available to academic laboratories through either Addgene (a non-profit plasmid distribution service; see http://www.addgene.org/zfc) or the Joung lab. All engineering and validation steps in the OPEN method are carried out in simple and inexpensive E. coli-based systems. Collectively, these reagents provide academics with an important platform which enables rapid engineering of ZFNs for zebrafish genes. When compared with ZFNs previously made to the kdr gene using a bacterial one-hybrid-based method, OPEN ZFNs were equally efficient at inducing mutations at their respective target sites but showed less toxicity and teratogenesis, even when larger amounts of ZFN-encoding RNA were injected. (Others have also noted the relatively greater toxicity of the kdr ZFNs compared with the golden and ntl ZFNs made using the Sangamo BioSciences zinc finger engineering technology [29,45] although we note that those comparisons were made between experiments which used different ZFN expression vectors.) The difference in toxicity we observed between the kdr ZFNs and our OPEN ZFNs is most likely due to the quality of the zinc finger arrays because all other sequences in the expression vectors are otherwise identical. One possible explanation for the greater toxicity of the kdr ZFNs is that the B1H system used to create them might permit identification of zinc finger arrays with lower specificities than those identified by the OPEN B2H system. Consistent with this, we note that one of the zinc finger arrays (ZFP2) used to make the kdr ZFNs failed to activate transcription in the B2H system (Table 1) and therefore would not have been identified as a positive clone if the kdr site had been targeted using the OPEN method. This result suggests that the B1H method used to engineer the kdr zinc finger arrays may be less stringent than the B2H-based OPEN approach, perhaps due to the use of a multi-copy reporter in the B1H system as opposed to the single-copy reporter used in the B2H system. This difference might reduce the selective pressure for DNA-binding specificity since the target DNA site in the B1H system will be present at a higher concentration in the bacterial cell relative to the ‘‘non- specific’’ DNA of the E. coli chromosome. An important priority for future work will be to examine the spectrum and range of ‘‘off- target’’ sites altered by OPEN ZFNs, perhaps using methods previously described by other groups [27,28]. The ability of OPEN to rapidly yield high quality ZFNs for a large number of different target sites should also improve prospects for using ZFNs to induce precise homologous recombination (HR) events at endogenous zebrafish genes. Repair of a ZFN-induced DSB by HR with an appropriately designed exogenous ‘‘donor template’’ (a process known as ZFN-induced gene targeting) has been used to introduce specific alterations or insertions with high efficiencies at endogenous genes in Drosophila [18,19], plant [Townsend et al, manuscript submitted], and human cells [23–26,43]. However, our experience using ZFNs in human cells suggests that ZFN-induced homologous recombination can be much more challenging to implement than ZFN-induced mutagenic NHEJ- mediated repair. For example, not all ZFNs that can induce NHEJ at their target sites will necessarily promote efficient HR (M. Maeder, S. Beganny, and J.K. Joung, unpublished results). Thus, the ability to use the OPEN method to rapidly engineer ZFNs with both high activities and low toxicities for many different target sites should greatly enhance prospects for successfully using ZFNs to induce specific HR events in zebrafish. Materials and Methods OPEN selections of zinc finger arrays Zinc finger arrays were selected using the OPEN method essentially as previously described [26] but with a small number of alterations that improve the speed and throughput of the protocol. We briefly summarize the overall protocol here with greater detail provided for steps of the protocol we altered for this report. A more detailed step-by-step protocol for performing OPEN selections is forthcoming (Maeder et al., manuscript in prepara- tion) and is currently available upon request from the Joung lab. To create recombinant libraries of zinc finger arrays for use in OPEN selections, zinc finger pools for target triplet subsites [26] were amplified by PCR using primers and conditions as previously described [26]. Amplified finger pool products were purified on 10% polyacrylamide gels and then fused together by PCR to create random combinations of three-finger arrays. These fusion PCR reactions were performed with equal concentrations of the three purified finger pool fragments and using primers and cycling conditions as previously described [26]. The resulting PCR product encoding a collection of three-finger arrays was purified on a 5% polyacrylamide gel and treated with Pfu polymerase and T4 polynucleotide kinase to create ligation-ready overhangs [26]. This fragment was then ligated to pBR-UV5-GP-FD2 vector that had been digested with the restriction enzyme BbsI. The resulting plasmids express the collection of zinc finger arrays as FLAG-tagged Gal11P fusions in the B2H system. Electroporation was then used to introduce these ligation products into E. coli XL-1 Blue cells and .36106 independent transformants were obtained for each library to ensure a minimum of three-fold oversampling of the theoretical library complexity of ,8.66105 (953). Libraries were then converted into infectious M13 phage as previously described [46]. OPEN selections were performed in two stages. In the first stage, an OPEN three-finger library was introduced by infection into a B2H selection strain harboring the full target DNA sequence of interest. For the selections described in this report, we grew selection strains as 1 ml cultures in 24-well, 10 ml-capacity PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4348