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 strateg...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2020-03-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-020-6655-4 |
| _version_ | 1819060130091630592 |
|---|---|
| author | Camilo Breton Peter M. Clark Lili Wang Jenny A. Greig James M. Wilson |
| author_facet | Camilo Breton Peter M. Clark Lili Wang Jenny A. Greig James M. Wilson |
| author_sort | Camilo Breton |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T14:22:06Z |
| format | Article |
| id | doaj.art-84dd2ff60759418d8a28d6e3f281d5ef |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-21T14:22:06Z |
| publishDate | 2020-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-84dd2ff60759418d8a28d6e3f281d5ef2022-12-21T19:00:45ZengBMCBMC Genomics1471-21642020-03-0121111210.1186/s12864-020-6655-4ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editingCamilo Breton0Peter M. Clark1Lili Wang2Jenny A. Greig3James M. Wilson4Gene Therapy Program, University of Pennsylvania Perelman School of MedicineGene Therapy Program, University of Pennsylvania Perelman School of MedicineGene Therapy Program, University of Pennsylvania Perelman School of MedicineGene Therapy Program, University of Pennsylvania Perelman School of MedicineGene Therapy Program, University of Pennsylvania Perelman School of MedicineAbstract 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.http://link.springer.com/article/10.1186/s12864-020-6655-4Genome editingOff-targetsNext-generation sequencingIn vivoEditingAAV integration |
| spellingShingle | Camilo Breton Peter M. Clark Lili Wang Jenny A. Greig James M. Wilson ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing BMC Genomics Genome editing Off-targets Next-generation sequencing In vivo Editing AAV integration |
| title | ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing |
| title_full | ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing |
| title_fullStr | ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing |
| title_full_unstemmed | ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing |
| title_short | ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing |
| title_sort | itr seq a next generation sequencing assay identifies genome wide dna editing sites in vivo following adeno associated viral vector mediated genome editing |
| topic | Genome editing Off-targets Next-generation sequencing In vivo Editing AAV integration |
| url | http://link.springer.com/article/10.1186/s12864-020-6655-4 |
| work_keys_str_mv | AT camilobreton itrseqanextgenerationsequencingassayidentifiesgenomewidednaeditingsitesinvivofollowingadenoassociatedviralvectormediatedgenomeediting AT petermclark itrseqanextgenerationsequencingassayidentifiesgenomewidednaeditingsitesinvivofollowingadenoassociatedviralvectormediatedgenomeediting AT liliwang itrseqanextgenerationsequencingassayidentifiesgenomewidednaeditingsitesinvivofollowingadenoassociatedviralvectormediatedgenomeediting AT jennyagreig itrseqanextgenerationsequencingassayidentifiesgenomewidednaeditingsitesinvivofollowingadenoassociatedviralvectormediatedgenomeediting AT jamesmwilson itrseqanextgenerationsequencingassayidentifiesgenomewidednaeditingsitesinvivofollowingadenoassociatedviralvectormediatedgenomeediting |