ITR-Seq, a Next-generation Sequencing Assay, Identifies Genome-wide DNA Editing Sites in Vivo Following Adeno-associated Viral Vector-mediated Genome Editing

Overview

Journal BMC Genomics

Publisher Biomed Central

Specialty Genetics

Date 2020 Mar 19

PMID 32183699

Citations 26

Authors

Camilo Breton

Peter M Clark

Lili Wang

Jenny A Greig

James M Wilson

Affiliations

Soon will be listed here.

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 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.

Citing Articles

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