Researchers describe the use of zinc finger nucleases (ZFNs) to achieve heritable gene disruption in zebrafish, overcoming previous limitations in targeted mutagenesis. ZFNs induce double-strand breaks in the genome, which are repaired to generate small insertions and deletions. The study targets the zebrafish golden (gol) and no tail/Brachyury (ntl) genes, resulting in loss-of-function phenotypes. ZFNs were designed to target specific loci, and their effectiveness was validated in zebrafish embryos. Disrupted ntl alleles were transmitted to over half of the adults tested at frequencies averaging 20%. The precision and frequency of gene disruption events suggest that ZFN technology can be widely applied to many organisms that allow mRNA delivery into the fertilized egg. Zebrafish are an excellent model for vertebrate development and disease due to their rapid development, transparent embryos, and ease of forward genetics. However, reverse genetic approaches in zebrafish are limited to mRNA knockdown strategies and TILLING for point mutations. Both methods have limitations, with morpholinos only affecting early development and TILLING being time-consuming and less effective for intron-rich genes. Conventional gene targeting in mouse embryonic stem cells requires cytotoxic drugs, which are not applicable in vertebrate embryos. ZFNs, initially developed to cleave DNA in vitro, are a fusion of the FokI cleavage domain and a DNA recognition domain with 3 or more C2H2 zinc finger motifs. Two ZFNs heterodimerize at a specific DNA position to induce a double-strand break, which is repaired via non-homologous end-joining (NHEJ) or homology-directed repair. NHEJ was used to disrupt genes in Drosophila and C. elegans, demonstrating the potential of ZFN technology in reverse genetic applications. The study used the gol and ntl genes as test loci to investigate the feasibility of using ZFNs for reverse-genetic applications in zebrafish. The gol gene encodes a transmembrane protein, and ZFNs were used to induce loss-of-function mutations. The ntl gene, a major regulator of early embryogenesis, was also targeted. ZFNs were designed to target specific exons, and their activity was tested in yeast-based assays. The results showed that ZFNs could induce targeted mutations in zebrafish embryos, leading to loss-of-function phenotypes. ZFNs were also tested for their ability to induce germline mutations. ZFN-injected fish were raised to sexual maturity and found to breed normally, with mutations transmitted to offspring. The frequency of germline mutations was around 20%, with over 60% of ntl ZFN-injected founders carrying mutations at the ntl locus. The study demonstrated that ZFNs can induce precise andResearchers describe the use of zinc finger nucleases (ZFNs) to achieve heritable gene disruption in zebrafish, overcoming previous limitations in targeted mutagenesis. ZFNs induce double-strand breaks in the genome, which are repaired to generate small insertions and deletions. The study targets the zebrafish golden (gol) and no tail/Brachyury (ntl) genes, resulting in loss-of-function phenotypes. ZFNs were designed to target specific loci, and their effectiveness was validated in zebrafish embryos. Disrupted ntl alleles were transmitted to over half of the adults tested at frequencies averaging 20%. The precision and frequency of gene disruption events suggest that ZFN technology can be widely applied to many organisms that allow mRNA delivery into the fertilized egg. Zebrafish are an excellent model for vertebrate development and disease due to their rapid development, transparent embryos, and ease of forward genetics. However, reverse genetic approaches in zebrafish are limited to mRNA knockdown strategies and TILLING for point mutations. Both methods have limitations, with morpholinos only affecting early development and TILLING being time-consuming and less effective for intron-rich genes. Conventional gene targeting in mouse embryonic stem cells requires cytotoxic drugs, which are not applicable in vertebrate embryos. ZFNs, initially developed to cleave DNA in vitro, are a fusion of the FokI cleavage domain and a DNA recognition domain with 3 or more C2H2 zinc finger motifs. Two ZFNs heterodimerize at a specific DNA position to induce a double-strand break, which is repaired via non-homologous end-joining (NHEJ) or homology-directed repair. NHEJ was used to disrupt genes in Drosophila and C. elegans, demonstrating the potential of ZFN technology in reverse genetic applications. The study used the gol and ntl genes as test loci to investigate the feasibility of using ZFNs for reverse-genetic applications in zebrafish. The gol gene encodes a transmembrane protein, and ZFNs were used to induce loss-of-function mutations. The ntl gene, a major regulator of early embryogenesis, was also targeted. ZFNs were designed to target specific exons, and their activity was tested in yeast-based assays. The results showed that ZFNs could induce targeted mutations in zebrafish embryos, leading to loss-of-function phenotypes. ZFNs were also tested for their ability to induce germline mutations. ZFN-injected fish were raised to sexual maturity and found to breed normally, with mutations transmitted to offspring. The frequency of germline mutations was around 20%, with over 60% of ntl ZFN-injected founders carrying mutations at the ntl locus. The study demonstrated that ZFNs can induce precise and