Rapid Mutation by OPEN ZFNs Figure 3. Frequencies and sequences of ZFN-induced mutations in somatic zebrafish cells. For each gene targeted by ZFNs, the wild-type sequence is shown at the top with ZFN binding sites marked. Deletions are indicated by grey highlighted red dashes and insertions by blue highlighted lower case blue letters. The number of times each wild-type mutant allele was isolated is shown in brackets. doi:10.1371/journal.pone.0004348.g003 Table 2. Frequencies of mutations from founder analysis Gene tfr2 dat telo hif1 aa # of fish screened # of mutant founder fish transmission rate for each mutant founder 12 17 8 2 4 1 2 1 1/10 (10%) 6/30 (20%) 1/11 (9%) 1/11 (9%) 1/10 (10%) 2/11 (18%) 6/10 (60%) 1/11 (9%) Numbers of fish screened for mutant founders are shown for each gene. For each mutant founder identified, the transmission rate is shown as the # of mutant embryos/# of embryos screened and as a percentage of mutant embryos identified (in parentheses). tfr2 = transferrin receptor 2; dat = dopamine transporter; telo = telomerase; hif1aa = hypoxia-inducible factor. doi:10.1371/journal.pone.0004348.t002 previous experience using OPEN to make ZFNs for endogenous human gene targets suggests that the success rate of this method is high but not 100% [26]. Assuming that a similar experience holds true in zebrafish, a prudent strategy might be to target more than one sequence in a gene of interest to improve the chances of successfully mutating that gene. Our analysis shows that most zebrafish gene transcripts possess multiple potential OPEN ZFN target sites and therefore suggests that a large number of genes will be targetable by such a strategy. As the academic community collectively gains experience using OPEN ZFNs in zebrafish (and other organisms), we anticipate that algorithms for identifying potential OPEN ZFN target sites will also continue to improve and evolve, thereby allowing for targeting of fewer sites per gene. Nonetheless, our results strongly suggest that OPEN in its current Figure 4. Sequences of ZFN-induced mutations transmitted through the germline. For each target gene, the wild-type sequence is shown at the top with ZFN binding sites marked and the mutated alleles found in founder progeny are shown below the wild-type sequence. Each mutant sequence shown was isolated from progeny of different founders. Deletions are indicated by grey highlighted red dashes and insertions by blue highlighted lower case blue letters. doi:10.1371/journal.pone.0004348.g004 PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4348