ITR-Seq successfully identified dose-dependent off-target nuclease activity in vivo, demonstrating higher sensitivity than GUIDE-Seq for detecting AAV integration sites following genome editing.
ITR-Seq enables unbiased, genome-wide identification of in vivo off-target editing sites following AAV-mediated gene therapy, providing a critical tool for assessing safety.
BACKGROUND: Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism's genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. RESULTS: Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. CONCLUSIONS: In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.
Breton et al. (Tue,) conducted a other in Genome editing off-target effects. ITR-Seq assay vs. GUIDE-Seq was evaluated on Identification of off-target nuclease activity in vivo. ITR-Seq successfully identified dose-dependent off-target nuclease activity in vivo, demonstrating higher sensitivity than GUIDE-Seq for detecting AAV integration sites following genome editing.