Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.
PLoS One. 2012;7(5):e37877. doi: 10.1371/journal.pone.0037877. Epub 2012 May 24.
Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.
锌指核酸酶（ZFNs）和转录激活因子样效应物核酸酶（TALENs）通过上下文相关组装（CoDA）和转录激活因子样效应物核酸酶（TALENs）提供了强大且用户友好的技术，可有效使斑马鱼中的基因失活。这些设计的核酸酶结合并在特定靶位点切割 DNA，诱导易错修复，导致插入或缺失突变。在这里，我们评估了这些技术在诱导嵌合体斑马鱼体细胞 DNA 突变方面的相对效率。我们发现，与 CoDA ZFNs 相比，TALENs 获得能够诱导突变的活性核酸酶的成功率更高。例如，测试的所有六个 TALEN 都在基因组靶位点诱导了 DNA 突变，而只有一部分 CoDA ZFNs 显示出可检测的诱变率。TALENs 的突变率也高于未经细菌双杂交测定预先筛选的 CoDA ZFNs，DNA 突变率范围为 20%-76.8%，而 CoDA ZFNs 的突变率为 1.1%-3.3%。此外，TALENs 的更广泛的靶向范围使我们能够在甲硫氨酸翻译起始位点诱导突变，这些序列使用 CoDA ZFN 平台无法靶向。TALENs 的毒性与 CoDA ZFNs 相似，>50%的注射动物存活至 3 天。综上所述，我们的结果表明，TALEN 技术为在斑马鱼中诱导靶向基因失活提供了一种强大的 CoDA ZFN 替代方法，使其成为在斑马鱼中创建靶向敲除突变体的首选技术。
