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

doi:10.13560/j.cnki.biotech.bull.1985.2016.01.035

Abstract

Abstract: 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(mtDNA)of TALENs. By means of pEGFP-N1 plasmid, the segments of target sequence were randomly integrated in the nuclear genome of HEK293F. 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.

Key words: transcription activator-like effector nucleases, unit assembly, mitochondrial DNA, HEK293F-T1

GAO Jing, WEI Di, CHI Zhen-fen, ZHANG Gui-rong, ZHANG Wan-ming, NIE Ling-yun. A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases[J]. Biotechnology Bulletin, 2016, 32(1): 220-228.

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