fragments from progeny that were resistant to BspHI were blunt-end cloned into the pCR4 Blunt-TOPO vector as described above and sequenced with the ‘‘T3 sequencing’’ primer. For the hifaa gene, we used primers OK.1934 and OK.1935 (Supplemental Table 26) to amplify the surrounding region by PCR and the resulting ,401 bp product was digested with the enzyme BfuAI. The PCR product from a wild-type allele will contain only one BfuAI site. Introduction of indel mutations at the ZFN target site will disrupt the BfuAI site thereby resulting in the generation of PCR products resistant to digestion by BfuAI. PCR fragments from progeny that were resistant to BfuAI were blunt-end cloned into the pCR4 Blunt-TOPO vector as described above and sequenced with the ‘‘T3 sequencing’’ primer. Identification of potential OPEN ZFN target sites in zebrafish transcripts ZFN target sites were generated from Danio rerio chromosomal contigs (Zv7) and gene table files (updated July 2008) from Ensemble (http://www.ensembl.org) for all mapped chromosomal protein coding transcripts. ZFN target sites were identified that can be targeted using currently available OPEN reagents [26] and that possess a spacer of 5, 6, or 7 nucleotides between the target half-sites. Only ZFN sites whose spacer falls entirely within an exon were identified as potential targets. In addition, because all previous 9 bp sites successfully targeted by OPEN to date have possessed at least one GNN triplet [26], we eliminated ZFN sites harboring one or more half-sites that are devoid of GNN triplets. Finally, because OPEN selections are performed in E. coli, ZFN sites containing either a dam or a dcm methylation site in either half-site were also eliminated from the target list. Supporting Information Table S1 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 1. Potential OPEN ZFN target sites within transcripts were identified as described in Materials and Methods. Gene ID and Transcript ID are from the Ensembl Danio rerio release 51 database. ‘‘Strand’’ indicates whether the ‘‘Target Site’’ shown (written 59 to 39) occurs on the forward (+) or reverse (2) strand. ‘‘ZFN Spacer Length’’ indicates the length of the spacer sequence located between the ZFN half-sites (5, 6, or 7 bps). ‘‘Coding Sequence Length’’ indicates the total nucleotide length of the coding sequence within the transcript and ‘‘ZFN Cleavage Site’’ indicates the nucleotide position of the cleavage site (i.e.–the first base of the ‘‘Target Site’’) within the coding sequence. Found at: doi:10.1371/journal.pone.0004348.s001 (2.22 MB XLS) Table S2 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 2. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s002 (2.39 MB XLS) Table S3 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 3. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s003 (2.28 MB XLS) Table S4 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 4. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s004 (2.05 MB XLS) Rapid Mutation by OPEN ZFNs Table S5 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 5. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s005 (3.12 MB XLS) Table S6 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 6. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s006 (2.28 MB XLS) Table S7 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 7. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s007 (2.80 MB XLS) Table S8 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 8. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s008 (2.44 MB XLS) Table S9 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 9. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s009 (2.21 MB XLS) Table S10 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 10. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s010 (1.78 MB XLS) Table S11 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 11. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s011 (1.75 MB XLS) Table S12 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 12. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s012 (1.55 MB XLS) Table S13 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 13. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s013 (1.98 MB XLS) Table S14 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 14. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s014 (1.62 MB XLS) Table S15 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 15. Data presented as described in the legend to Table S1. Found at: doi:10.1371/journal.pone.0004348.s015 (1.84 MB XLS) Table S16 Potential OPEN ZFN target sites in gene transcripts encoded on zebrafish chromosome 16. Data presented as described in the legend to Table S1. PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4348