Transposition has a key role in reshaping genomes of all living organisms¹. Insertion sequences of IS200/IS605 and IS607 families² are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established^3,4,5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases^6,7,8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR–Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

转座在重塑所有生物体的基因组方面发挥着关键作用¹ 。 IS200/IS605 和 IS607 家族²的插入序列属于最简单的可移动遗传元件，仅包含其转座及其调节所需的基因。这些元件编码tnpA转座酶，这对于动员是必需的，并且通常携带辅助tnpB基因，该基因对于转座来说是可有可无的。尽管 TnpA 在 IS200/IS605 转座子动员中的作用已有充分记录，但 TnpB 的功能仍然很大程度上未知。有人认为 TnpB 在转座调节中发挥作用，尽管尚未建立这种机制^3,4,5 。生物信息学分析表明，TnpB 可能是 CRISPR–Cas9/Cas12 核酸酶的前身^6,7,8 。然而，尚未发现 TnpB 具有生化活性。在这里，我们证明耐辐射奇球菌ISDra2 的 TnpB 是一种 RNA 指导的核酸酶，它由来自转座子右端元件的 RNA 引导，切割 5'-TTGAT 转座子相关基序旁边的 DNA。我们还表明，TnpB 可以被重新编程以切割人类细胞中的 DNA 靶位点。总之，这项研究通过强调 TnpB 在转座中的作用，扩展了我们对转座机制的理解，通过实验证实了 TnpB 是 CRISPR-Cas 核酸酶的功能祖细胞，并将 TnpB 确立为基因组编辑新系统的原型。