Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model

作者：Zhifang Li, Ruochen Guo, Xiao-Zhi Sun, Guoling Li, Zhuang Shao, Xiaona Huo, Rongrong Yang, Xinyu Liu, Xi Cao, Hainan Zhang, Weihong Zhang, Xiaoyin Zhang, Shuangyu Ma, Meiling Zhang, Yuanhua Liu, Yinan Yao, Jinqi Shi, Hui Yang, Chunyi Hu, Yingsi Zhou, Chunlong Xu · 发表于：Nature Communications · 年份：2024 · DOI：10.1038/s41467-024-45197-z · 被引用次数：44 · 研究领域：CRISPR and Genetic Engineering、RNA regulation and disease、Virus-based gene therapy research

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.