Liu Yu, Weng Zhixin, Zhai Ziheng, Xu Zhengmeng, Liu Xiaojing, Qiang Huanran, Tian Kun, Rao Bingcheng, Zhou Guohua, Guo Yongjian, Xu Shu
School of Basic Medical Science and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China.
Department of Anesthesiology, Perioperative and Pain Medicine, Nanjing First Hospital, China Pharmaceutical University, Nanjing, Jiangsu 210006, China.
Nucleic Acids Res. 2025 Jul 19;53(14). doi: 10.1093/nar/gkaf723.
A DNA editing tool TaqTth-hpRNA was developed in this study, composed of a compact recombinant TaqTth nuclease (832 aa) and a simple hairpin-RNA guiding probe (hpRNA). In vitro biochemical studies showed the TaqTth-hpRNA efficiently cleaves artificially synthesized single-stranded DNA without stringent sequence motif like protospacer adjacent motif (PAM). It can also cleave the genomic DNA of Escherichia coli with ∼80% efficiency. The TaqTth-hpRNA cleavage of genomic DNA in mammalian cells generated products with large fragment deletions mediated by the microhomology-mediated end joining pathway. In addition, the cleavage was sensitive to mismatches in targeted regions, which was applied to specific damage of the APPlon mutation in Alzheimer's disease without disrupting the APPwt locus. It is worth mentioning that the APPlon sequence has only one base difference from that of APPwt. The characteristics of small size, no PAM requirement, high specificity, and large deletion products make the TaqTth-hpRNA a potential therapeutic strategy for treating autosomal dominant disorders in the future.
本研究开发了一种DNA编辑工具TaqTth-hpRNA，它由一个紧凑的重组TaqTth核酸酶（832个氨基酸）和一个简单的发夹RNA引导探针（hpRNA）组成。体外生化研究表明，TaqTth-hpRNA能有效切割人工合成的单链DNA，而无需像原间隔相邻基序（PAM）那样严格的序列基序。它还能以约80%的效率切割大肠杆菌的基因组DNA。TaqTth-hpRNA对哺乳动物细胞基因组DNA的切割产生了由微同源性介导的末端连接途径介导的大片段缺失产物。此外，这种切割对靶向区域的错配敏感，可用于阿尔茨海默病中APP lon突变的特异性损伤，而不破坏APP wt基因座。值得一提的是，APP lon序列与APP wt序列只有一个碱基差异。TaqTth-hpRNA体积小、无需PAM、特异性高以及产生大片段缺失产物的特点，使其成为未来治疗常染色体显性疾病的一种潜在治疗策略。
