Liu Yun, Luo Daji, Lei Yong, Hu Wei, Zhao Hui, Cheng Christopher H K
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China; School of Biomedical Sciences Core Laboratory, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China; Department of Genetics, School of Basic Medical Sciences, Wuhan University, Wuhan, China.
Methods. 2014 Aug 15;69(1):58-66. doi: 10.1016/j.ymeth.2014.02.011. Epub 2014 Feb 17.
Transcription activator like effector nucleases (TALENs) is a promising approach to disrupt intended genomic loci. The assembly of highly effective TALENs is critical for successful genome editing. Recently we reported a convenient and robust platform to construct customized TALENs. The TALENs generated by this platform have been proven to be highly effective for gene disruption in Xenopus tropicalis and zebrafish as well as large genomic deletions in zebrafish. The one-time success rate of targeted gene disruption is about 90% for more than 100 genomic loci tested, with the mutation frequencies often reaching above 50%. Here we describe the validated protocol for TALEN assembly, methods for generating gene knockout animals in X. tropicalis and zebrafish, as well as the protocol for engineering large genomic deletions in zebrafish.
转录激活样效应因子核酸酶（TALENs）是一种用于破坏特定基因组位点的有前景的方法。高效TALENs的组装对于成功的基因组编辑至关重要。最近，我们报道了一个构建定制化TALENs的便捷且强大的平台。该平台产生的TALENs已被证明在热带爪蟾和斑马鱼中对基因破坏以及在斑马鱼中对大片段基因组缺失非常有效。对于测试的100多个基因组位点，靶向基因破坏的一次性成功率约为90%，突变频率通常达到50%以上。在此，我们描述了经过验证的TALEN组装方案、在热带爪蟾和斑马鱼中产生基因敲除动物的方法，以及在斑马鱼中构建大片段基因组缺失的方案。
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