5.52 5.23 6.03 5.63 6.57 6.76 3.44 3.60 1.20 3.04 3.23 2.82 6.54 10.24 9.93 10.98 8.84 7.87 Rapid Mutation by OPEN ZFNs 0.66 0.26 1.78 0.42 0.12 0.75 0.33 0.29 0.15 0.11 0.16 0.14 1.54 0.75 0.09 1.62 0.88 2.44 Table 1. cont. OZ489 OZ490 OZ491 OZ492 OZ493 OZ494 OZ495 OZ496 OZ497 OZ498 OZ499 OZ500 OZ501 OZ502 OZ503 OZ504 OZ505 OZ506 TfR2_2R TfR2_2R TfR2_2R TfR2_2R TfR2_2R TfR2_2R Grck_5L Grck_5L Grck_5L Grck_5L Grck_5L Grck_5L Grck_5R Grck_5R Grck_5R Grck_5R Grck_5R Grck_5R QRQALDR QRQALDR SAQALAR QRQALDR QRQALDR QRQALDR GGAa QQAHLVR QQAHLVR DNAHLAR QQAHLVR QQAHLVR QQAHLVR GAGc KHSNLTR KHSNLTR KHSNLTR KHSNLTR KHSNLAR KHSNLTR QQTNLTR QATNLQR QQTNLAR QSANLSR QGGNLTR QQTNLTR GCA QAETLKR QAETLKR QGETLKR QTETLKR QTETLKR QNETLRR GCA QTETLKR QKETLNR QKETLNR QMETLKR QRETLKR QRETLKR VGGNLAR VGSNLTR VGSNLTR VGSNLTR VGGNLSR VGSNLTR GCA QTATLKR QTATLKR QGNSLNR QTATLKR QTATLKR QTATLKR GCA QTATLKR QPNTLTR QPNTLTR QGGTLRR QGGTLVR QGGTLRR ZFN Name Site Name F1 subsite/RH sequence F2 subsite/RH sequence F3 subsite/RH sequence Mean B2H fold-activation S.D. of B2H fold-activation Each OPEN zinc finger array was assigned an OZ___ designation which permits their unique identification in the web-based Zinc Finger Database (ZiFDB) program [47]. Previously published zinc finger arrays targeted to the kdr gene (isolated by B1H selection) [27] are also shown. Each nine bp target site was named as follows: ‘‘gene name or abbreviation’’, ’’exon number’’, and ‘‘L’’ or ‘‘R’’ indicating left or right half-site. The amino acids selected in the three zinc finger recognition helices of each array are shown (residues are shown left to right in the order 21, 1, 2, 3, 4, 5, 6 numbered relative to the helix start). B2H values that fall below the cut-off of three-fold activation in the B2H system are italicized. The names of zinc finger arrays tested as ZFNs in zebrafish are shown in bold italics. Abbreviations key: DT = dopamine transporter, HIF = hif1aa, Telo = telomerase, TfR2 = transferrin receptor 2, and Grck = gridlock. doi:10.1371/journal.pone.0004348.t001 mutagenesis efficiency comparable to that previously observed by Wolfe and Lawson [27]. Having validated our ZFN expression vectors and mutation detection assay, we next tested each of the five ZFN pairs we made by OPEN in zebrafish embryos. Interestingly, with our OPEN ZFNs, we were able to inject four-times or more RNA per embryo (400 to 660 pg of RNA/embryo) compared with the Wolfe/ Lawson kdr ZFNs and yet observed comparable or sometimes lower rates of death/monster formation (Figure 2). When OPEN ZFNs were injected at 100–200 pg of RNA/embryo, death/ monster rates were even lower (0–27%; data not shown). To check for evidence of mutations in somatic cells, we isolated pooled genomic DNA from 4–10 embryos for each pair of ZFNs and performed limited cycle PCR/DNA sequencing to assess whether mutations were introduced at their intended endogenous gene targets. As shown in Figures 3B–3F, we observed insertion or deletions at the ZFN cleavage site for all five endogenous genes with mutagenesis rates ranging from 3%–20%. Nearly all of these indel mutations are predicted to create frameshift mutations although a few frame-preserved mutations are also observed. We conclude that OPEN ZFNs can efficiently induce mutations at endogenous genes in somatic zebrafish cells. Efficient germline transmission of mutations induced by OPEN ZFNs We tested whether ZFN-induced mutations observed in somatic zebrafish cells could be transmitted efficiently through the germline. Injected embryos remaining from four of the five somatic cell experiments described above were allowed to mature to adulthood and crossed with wild-type fish (fish in which gridlock had been targeted have not yet reached maturity and therefore have not yet been tested). To identify founders, we analyzed individual embryos from these crosses using either direct DNA sequencing or a restriction digest assay that checks for the loss of a restriction site located at the ZFN-induced DSB (see Materials and Methods). As shown in Table 2, founders were identified at frequencies of ,6%, 33%, 25%, and 50% for mutations in the dat, tfr2, telomerase and hif1aa genes, respectively. The percentages of embryos harboring ZFN-induced mutations from founders ranged from 9% to 60% (Table 2). We sequenced the mutations from a subset of these embryos to determine the molecular nature of the indels and found both frame-shifted and frame-preserved mutations (Figure 4). We conclude that mutations generated by OPEN ZFNs undergo efficient germline transmission in zebrafish. In silico identification of OPEN ZFN targets within endogenous zebrafish genes Using a program similar to the web-based software program ZiFiT v3.0 [26,44], we searched for sites in endogenous zebrafish genes that could potentially be targeted by OPEN (see Materials and Methods). To do this, we searched the first three coding exons of 29,291 protein-coding gene transcripts that have known mapped chromosomal locations and that are present in the Ensembl Danio rerio database (release 51; http://www.ensembl. PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4348