A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expres...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2019-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050118301219 |
| _version_ | 1818175624694464512 |
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| author | Ang Li Ciaran M. Lee Ayrea E. Hurley Kelsey E. Jarrett Marco De Giorgi Weiqi Lu Karol S. Balderrama Alexandria M. Doerfler Harshavardhan Deshmukh Anirban Ray Gang Bao William R. Lagor |
| author_facet | Ang Li Ciaran M. Lee Ayrea E. Hurley Kelsey E. Jarrett Marco De Giorgi Weiqi Lu Karol S. Balderrama Alexandria M. Doerfler Harshavardhan Deshmukh Anirban Ray Gang Bao William R. Lagor |
| author_sort | Ang Li |
| collection | DOAJ |
| description | Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapy |
| first_indexed | 2024-12-11T20:03:15Z |
| format | Article |
| id | doaj.art-312deba985e94bf79a24b835737e75c8 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-11T20:03:15Z |
| publishDate | 2019-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-312deba985e94bf79a24b835737e75c82022-12-22T00:52:28ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012019-03-0112111122A Self-Deleting AAV-CRISPR System for In Vivo Genome EditingAng Li0Ciaran M. Lee1Ayrea E. Hurley2Kelsey E. Jarrett3Marco De Giorgi4Weiqi Lu5Karol S. Balderrama6Alexandria M. Doerfler7Harshavardhan Deshmukh8Anirban Ray9Gang Bao10William R. Lagor11Department of Bioengineering, Rice University, Houston, TX 77030, USADepartment of Bioengineering, Rice University, Houston, TX 77030, USADepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USADepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA; Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, TX 77030, USADepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USADepartment of Bioengineering, Rice University, Houston, TX 77030, USADepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USADepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USADepartment of Bioengineering, Rice University, Houston, TX 77030, USADepartment of Bioengineering, Rice University, Houston, TX 77030, USADepartment of Bioengineering, Rice University, Houston, TX 77030, USA; Corresponding author: Gang Bao, Department of Bioengineering, Rice University, Houston, TX 77030, USA.Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA; Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, TX 77030, USA; Corresponding author: William R. Lagor, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans. Keywords: CRISPR/Cas9, adeno-associated virus, AAV, in vivo delivery, self-deleting, somatic genome editing, liver, AAV-CRISPR, gene therapyhttp://www.sciencedirect.com/science/article/pii/S2329050118301219 |
| spellingShingle | Ang Li Ciaran M. Lee Ayrea E. Hurley Kelsey E. Jarrett Marco De Giorgi Weiqi Lu Karol S. Balderrama Alexandria M. Doerfler Harshavardhan Deshmukh Anirban Ray Gang Bao William R. Lagor A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing Molecular Therapy: Methods & Clinical Development |
| title | A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing |
| title_full | A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing |
| title_fullStr | A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing |
| title_full_unstemmed | A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing |
| title_short | A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing |
| title_sort | self deleting aav crispr system for in vivo genome editing |
| url | http://www.sciencedirect.com/science/article/pii/S2329050118301219 |
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