Hosoda Ayako, Nakazato Issei, Okuno Miki, Itoh Takehiko, Takanashi Hideki, Tsutsumi Nobuhiro, Arimura Shin-Ichi
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo 113-8657, Japan.
Research Fellow of Japan Society for the Promotion of Science, Tokyo 102-0083, Japan.
Plant Biotechnol (Tokyo). 2024 Dec 25;41(4):357-365. doi: 10.5511/plantbiotechnology.24.0510a.
Recently a cytidine deaminase-based method for highly efficient C-to-T targeted base editing was developed and has been used with CRISPR-mediated systems. It is a powerful method for genome engineering, although it is prone to off-target effects and has a limited targeting scope. Transcription activator-like effector (TALE)-based tools which allow longer recognition sequences than do CRISPR/Cas9 systems, can also be used for targeted C-to-T base editing. Here, we describe a method that efficiently achieved targeted C-to-T substitutions in nuclear genes using cytidine deaminase fused to a TALE DNA-binding domain. We used a single pair of TALEs with a novel TALE-repeat unit that can recognize all four DNA bases, especially to allow for variations in the third base of codons in homologous genes. This targeting strategy makes it possible to simultaneously base edit almost identical sites in multiple isoforms of a gene while suppressing off-target substitutions.
最近，一种基于胞苷脱氨酶的高效C到T靶向碱基编辑方法被开发出来，并已用于CRISPR介导的系统。这是一种用于基因组工程的强大方法，尽管它容易产生脱靶效应且靶向范围有限。基于转录激活样效应物（TALE）的工具比CRISPR/Cas9系统允许更长的识别序列，也可用于靶向C到T碱基编辑。在这里，我们描述了一种方法，该方法使用与TALE DNA结合结构域融合的胞苷脱氨酶在核基因中高效实现靶向C到T替换。我们使用了一对带有新型TALE重复单元的TALE，该单元可以识别所有四种DNA碱基，特别是允许同源基因密码子第三个碱基的变异。这种靶向策略使得在抑制脱靶替换的同时，能够对基因的多个异构体中的几乎相同位点进行同时碱基编辑成为可能。
