Department of Biochemistry and Molecular Biology, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China.
Biotechnol Lett. 2014 Mar;36(3):471-9. doi: 10.1007/s10529-013-1387-z. Epub 2013 Oct 25.
Transcription activator-like effector nucleases (TALENs) have emerged as a newly developed approach for genome editing. However, its application in targeting specific genomic loci susceptible to DNA damage remains obscure. Here, we report a modified approach for TALENs-based targeting of FATS, a fragile-site gene whose major introns have AT-rich sequence and di-nucleotide repeats. Two pairs of FATS-TALENs were designed to cleave two sites specifically at a coding exon of FATS. After in vitro transcription, the mRNA from FATS-TALEN pairs was microinjected into mouse zygotes. The targeting efficiency of two FATS-TALEN pairs in vivo was more than threefold higher than that of one FATS-TALEN pair. Moreover, large-size DNA deletions were detected, which were heritable and easily detectable by PCR. Our study indicates that the double-hit TALEN approach enhances targeting efficiency in vivo and provides convenience for monitoring germline transmission of mutations by PCR, which will facilitate the functional research on fragile-site genes.
转录激活样效应物核酸酶（TALENs）已成为一种新兴的基因组编辑方法。然而，其在靶向易受 DNA 损伤的特定基因组位点方面的应用仍不清楚。在这里，我们报告了一种改良的方法，用于基于 TALENs 靶向 FATS，这是一个脆性位点基因，其主要内含子具有富含 AT 的序列和二核苷酸重复。设计了两对 FATS-TALEN 来特异性地切割 FATS 编码外显子的两个位点。体外转录后，将来自 FATS-TALEN 对的 mRNA 微注射到小鼠受精卵中。在体内，两对 FATS-TALEN 对的靶向效率比一对 FATS-TALEN 对高三倍以上。此外，还检测到了可遗传且易于通过 PCR 检测到的大片段 DNA 缺失。我们的研究表明，双击中的 TALEN 方法可提高体内靶向效率，并通过 PCR 方便地监测基因突变的种系传递，这将有助于脆性位点基因的功能研究。
