Abstract
BackgroundIdentifying 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.ResultsHere, 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.ConclusionsIn 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.
Highlights
Identifying nuclease-induced double-stranded breaks in Deoxyribonucleic acid (DNA) on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies
Developing the inverted terminal repeat (ITR)-Seq assay to evaluate meganuclease activity in non-human primates We sought to develop a methodology for unbiased, genome-wide identification of sites of ITR integration
Based on a peak in absolute frequency at position 82 of the AAV2 reference genome (Fig. 1a), we determined that the most frequent base position of ITR integration occurs 5′ upstream of the Rep-binding element. We used this information to design an ITR-specific primer that hybridizes 5′ upstream of the observed ITR-integration start site. We used this primer in a novel next-generation sequencing (NGS) assay, based on anchored multiplexed Polymerase chain reaction (PCR), to identify the ITR-genomic DNA junction following insertional mutagenesis (Fig. 1c)
Summary
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. The host cell subsequently repairs these DSBs, resulting in edited alleles that can contain insertions and deletions (indels) or more complex genomic rearrangements [1]. The specificity of these nucleases is conferred by either 1) a single-guide RNA (sgRNA) in the case of the CRISPRCas system [2]; 2) a rational design and evolution of sequence-specific DNA-binding domains fused to a nuclease (e.g., TALENs or zinc fingers [3]); or 3) engineered versions of restriction enzymes (e.g., meganucleases) [4, 5]. During the preparation of this manuscript, Hanlon et al published an assay, similar to ours, to identify AAV integration after CRISPR-Cas9-mediated editing to identify onand off-target sites [15]
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