Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination^1,2,3,4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. ^5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR–Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans⁷. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility⁸, phage susceptibility⁹, and host immunity¹⁰. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

转座子编码的 tnpB 和 iscB 基因编码 RNA 引导的 DNA 核酸酶，通过定向 DNA 切割和同源重组促进自身的自私扩散 ^1,2,3,4 。这些广泛存在的基因家族在进化时间尺度上被反复驯化，导致多种 CRISPR 相关核酸酶的出现，包括 Cas9 和 Cas12（参考文献 ^5,6 ）。我们着手测试这样一个假设：TnpB 核酸酶可能也被重新用于 CRISPR-Cas 适应性免疫之外的新的、意想不到的功能。在这里，我们利用系统发生学、结构预测、比较基因组学和功能分析，揭示了可编程转录因子的多个独立发生事件，我们将其命名为 TnpB 样核酸酶死亡阻遏物 (TldR)。这些蛋白质使用天然存在的引导 RNA 特异性靶向基因组的保守启动子区域，以类似于人类发明的 CRISPR 干扰技术的机制产生有效的基因抑制 ⁷ 。着眼于肠杆菌科中广泛发现的 TldR 进化枝，我们发现噬菌体利用 TldR 和相邻编码的噬菌体基因的联合作用来改变宿主鞭毛组件的表达和组成，这是一种可能影响运动性的转变 ⁸ 和宿主免疫 ¹⁰ 。总的来说，这项工作展示了通过转座子编码基因的重复外展而实现的多种分子创新，并揭示了多种 RNA 引导转录因子的进化轨迹。