ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing

Abstract

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 vectordelivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genomeediting nucleases in animal models and clinical studies. This information can help enhance the safety profile of geneediting therapies.

Targeted Gene Delivery: Where to Land

“…First, targeted integration can lower serum albumin levels (Zhang et al, 2019 ; Ou et al, 2020 ) and albumin mutations have been observed in human hepatocellular carcinoma (Cancer Genome Atlas Research Network, 2017 ; Rao et al, 2017 ). Second, long-term AAV-mediated expression of endonucleases can result in off-target editing and unwanted AAV insertions (Li et al, 2019 ; Breton et al, 2020 ; Wang et al, 2020c ). Finally, pre-existing liver conditions and immune responses against AAV vectors used to deliver transgenes or nucleases severely limit the number of eligible patients (Boutin et al, 2010 ; Simhadri et al, 2018 ).…”

CRISPR-Cas9-Mediated In Vivo Gene Integration at the Albumin Locus Recovers Hemostasis in Neonatal and Adult Hemophilia B Mice

“…Deep sequencing could be a powerful method for validated measurement of AAV integration frequencies. 42,43 We found different parts of the AAV vector sequences inserted into DSBs in our previous work. 6 Due to the limitation of amplicon sequencing method in this study, we could not determine the extent of vector sequence integration around target region.…”

The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers

“…An HTS‐based assay was recently developed to identify off‐target nuclease activity after the AAV‐mediated genome edition in vivo. [ 32 ] In that protocol, named ITR‐Seq, PCR, and adapter optimizations are performed to specifically amplify ITR‐genomic DNA junctions. Combining multiple sequencing technologies could provide complementary information and reduce the risks associated with the inherent technical errors of each platform.…”