Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage

• 成果类型: Article
• 署名作者: Liu, Zi-Xian; Zhang, Shouyue; Zhu, Han-Zhou; Chen, Zhi-Hang; Yang, Yun; Li, Long-Qi; Lei, Yuan; Liu, Yun; Li, Dan-Yuan; Sun, Ao; Li, Cheng-Ping; Tan, Shun-Qing; Wang, Gao-Li; Shen, Jie-Yi; Jin, Shuai; Gao, Caixia; Liu, Jun-Jie Gogo
• 署名单位: Tsinghua University; Chinese Academy of Sciences; Institute of Genetics & Developmental Biology, CAS; Chinese Academy of Sciences; University of Chinese Academy of Sciences, CAS
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-12679
• DOI: 10.1126/science.adh4859
• 发表日期: 2024-02-02
• 页码: 495-+
• 关键词: group-ii intron; self-splicing rna; gene conversion; escherichia-coli; orf-less; RECOGNITION; replication; mutations; mechanism; insights

摘要

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.