Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance¹. IS200- and IS605-family transposons undergo ‘peel-and-paste’ transposition catalysed by a TnpA transposase², but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively^3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases^5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR–Cas adaptive immunity for antiviral defence.

插入序列是细菌中发现的紧凑且普遍的转座元件，仅编码其动员和维持所需的基因¹。^{IS200 和 IS605 家族转座子经历 TnpA 转座酶2}催化的“剥离和粘贴”转座，但它们也编码多种 TnpB 和 IscB 家族蛋白，这些蛋白在进化上与 CRISPR 相关效应子 Cas12 和 Cas9 相关，分别是^3，4。最近的研究表明，TnpB 和 IscB 作为 RNA 引导的 DNA 核酸内切酶发挥作用^5,6，但这种活性的更广泛的生物学作用仍然是个谜。在这里，我们证明 TnpB 和 IscB 对于防止 TnpA 转座机制导致的永久性转座子丢失至关重要。我们从嗜热脂肪地芽孢杆菌中选择了一个相关插入序列家族，它们编码多个 TnpB 和 IscB 直系同源物，并表明单个 TnpA 转座酶对转座子动员具有广泛的活性。转座子侧翼序列重新连接后形成的供体接头被 RNA 引导的 TnpB 和 IscB 核酸酶有效地靶向切割，并且与单独表达 TnpA 的条件相比，TnpB 和 TnpA 的共表达导致转座子保留显着增加。值得注意的是，TnpA 和 TnpB 也刺激了重组频率，超过了单独使用 TnpB 时观察到的重组频率。总的来说，这项研究揭示了RNA引导的DNA切割作为一种原始生化活动而出现，以偏向转座元件的自私遗传和传播，后来在用于抗病毒防御的CRISPR-Cas适应性免疫的进化过程中被采用。