Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

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 2,500 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. To fully appreciate L1 insertions, we first employed the improved amplicon sequencing at multiple target sites for canonical CRISPR/Cas9 system and multiple advanced genome editing technologies such as prime editors (PE) enabling precise base editing including PE2, PE3 and PE3b system, cytosine base editors (CBEs) including BE2, BE3, BE4-Gam and AncBE4max, as well as adenine base editors (ABE8e) system. We also performed polyA L1 insertion location sequencing developed based on HTGTS as described previously (Chiarle et al., 2011), the polyA approach is particularly designed for capturing the canonical TPRT L1 retrotransposition.