TALENs新型模块组装法及活性鉴定方法

A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases

旨在提供一种新型的转录激活样效应子核酸酶(Transcription activator-like effector nucleases,TALENs)模块组装法(Unit assembly,UA)及活性鉴定方法。利用TALE-NT 2.0在线工具在线粒体DNA(Mitochondrial DNA,mt DNA)设计TALENs识别位点和剪切位点,借助p EGFP-N1质粒将该段靶序列随机整合于HEK293F核基因组中,构建HEK293F-T1细胞系,用于TALENs活性鉴定。依据转录激活样效应子(Transcription activator-like effectors,TALEs)自然单元模块序列,设计出一种新型的人工TALEs偏单元;根据TALENs识别位点,选取相应一单元模块,利用UA法组装;并设计出含有相应双酶切位点的TALEs串联模块序列和TALENs载体序列,定向连接后瞬时转染HEK293F-T1细胞系。结果显示,新型模块组装法定向组装TALEs模块,克隆效率高且瞬时转染后可看到清晰的套峰。结果表明,新型模块组装方法提高了TALENs构建过程中的克隆效率,并且不受末位0.5模块的RVD(repeat-variable diresidue)限制,增加了靶序列设计的灵活性。

A novel method of unit assembly（UA）and activity assay based on transcription activator-like effector nucleases（TALENs）are described. An online tool TALE-NT 2.0 was used to design recognition and splice sites on mitochondrial DNA（mt DNA）of TALENs. By means of p EGFP-N1 plasmid, the segments of target sequence were randomly integrated in the nuclear genome of HEK293 F. The constructed cell line HEK293F-T1 was for activity assay of TALENs. Based on transcription activator-like effector（TALE）natural repeats, a new type of artificial TALEs single-unit repeats were designed. According to the recognition sites of TALENs, appropriate single-unit repeats were selected and assembled by UA. TALEs series unit sequence containing the corresponding double enzyme cutting sites and TALENs vector sequence were designed, which was directionally ligated and transiently transfected into HEK293F-T1 cell line. The results showed that the TALES units were directionally assembled by new method, and it had a high cloning efficiency;moreover, the nested peaks clearly appeared after transient transfection. In conclusion, the novel method of unit assembly improves cloning efficiency of constructing TALLENs, even is not limited by repeat-variable residue（RVD）in the last 0.5 unit, and increases the flexibility of designing the target sequences.