CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

基于 CRISPR/Cas9 的基因组编辑彻底改变了实验分子生物学，并进入了靶向基因治疗的临床世界。识别发生在 CRISPR/Cas9 目标位点的 DNA 修饰对于确定编辑工具的效率和安全性至关重要。在这里，我们表明 LINE-1 (L1) 逆转录转座子的插入可以频繁发生在 CRISPR/Cas9 编辑位点。结合 PolyA-seq 和改进的扩增子测序，我们在 HEK293T、HeLa 和 U2OS 细胞的多个 CRISPR/Cas9 编辑位点表征了超过 2500 个从头 L1 插入。这些 L1 逆转录转座事件利用 CRISPR/Cas9 诱导的 DSB 形成并需要 L1 RT 活性。重要的是，在由主要编辑器 (PE)、胞苷或腺嘌呤碱基编辑器（CBE 或 ABE）进行的基因组编辑过程中，从头插入 L1 很少见，这与它们减少的 DSB 形成一致。