CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.

CRISPR-Cas系统和IS200/IS605转座子相关的TnpB已被用于基因组编辑技术的开发。通过生物信息学分析和生化实验，我们提出了一个新的 RNA 引导 DNA 核酸内切酶家族。我们的生物信息学分析初步确定了保守的 RAGATH-18 衍生 RNA (reRNA) 及其上游 IS607 TnpB 的稳定共现，平均长度为 390 个氨基酸。 IS607 TnpB 通过与 reRNA 相互作用形成可编程 DNA 酶。我们发现 IS607 TnpB 系统在细菌和人类细胞中具有强大的 dsDNA 干扰活性。厚壁菌门细菌IS607 TnpB 系统 (ISFba1 TnpB) 的进一步表征表明，其 dsDNA 切割活性对人类细胞中指导序列和目标序列之间的单个错配非常敏感。我们的研究结果表明，reRNA 引导序列中 20 nt 的长度可实现 ISFba1 TnpB 的最高 DNA 切割活性。 ISFba1 TnpB 效应蛋白与其同源 RAGATH-18 基序的 reRNA 和 dsDNA 靶标结合的冷冻电镜结构揭示了 ISFba1 TnpB 识别 reRNA 的机制、reRNA 引导的 dsDNA 靶向以及 ISFba1 TnpB 系统的敏感性指导 DNA 和目标 DNA 之间的碱基不匹配。总的来说，这项研究确定了 IS607 TnpB 家族的紧凑和特异性 RNA 引导的 DNA 酶，在基因编辑中具有巨大的应用潜力。