In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system
Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises conce...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050121000206 |
| _version_ | 1818557822044995584 |
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| author | Qian Li Jing Su Yi Liu Xiu Jin Xiaomei Zhong Li Mo Qingnan Wang Hongxin Deng Yang Yang |
| author_facet | Qian Li Jing Su Yi Liu Xiu Jin Xiaomei Zhong Li Mo Qingnan Wang Hongxin Deng Yang Yang |
| author_sort | Qian Li |
| collection | DOAJ |
| description | Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises concerns with specificity and immunogenicity. Thus, it would be advantageous to limit the duration of Cas9 expression following delivery. In this study, we have engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, which consists of a Cas9 nuclease from Staphylococcus aureus (SaCas9), a chimeric single guide RNA (sgRNA) molecule targeting PCSK9, and flanking sites targeted by this sgRNA. The self-cleavage system generated a negative feedback loop where Cas9 cut both the target genomic locus and the AAV vector, thus self-limiting the expression of Cas9. We demonstrated that this system could reduce ∼60% expression of SaCas9 protein and had a 20-fold reduction in off-target activity at 24 weeks post-vector administration in vivo. Moreover, the on-target editing efficacy was not compromised and resulted in a stable reduction in circulating PCSK9 and serum cholesterol. The inclusion of this self-cleavage system in gene-editing approaches could increase the safety profile of AAV-delivered genome-editing nucleases and thereby promote its clinical transformation. |
| first_indexed | 2024-12-14T00:04:38Z |
| format | Article |
| id | doaj.art-44c68dde0c7746e19a372bda5ec90f68 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-14T00:04:38Z |
| publishDate | 2021-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-44c68dde0c7746e19a372bda5ec90f682022-12-21T23:26:06ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012021-03-0120652659In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR systemQian Li0Jing Su1Yi Liu2Xiu Jin3Xiaomei Zhong4Li Mo5Qingnan Wang6Hongxin Deng7Yang Yang8State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, ChinaState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China; Corresponding author: Yang Yang, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China.Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises concerns with specificity and immunogenicity. Thus, it would be advantageous to limit the duration of Cas9 expression following delivery. In this study, we have engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, which consists of a Cas9 nuclease from Staphylococcus aureus (SaCas9), a chimeric single guide RNA (sgRNA) molecule targeting PCSK9, and flanking sites targeted by this sgRNA. The self-cleavage system generated a negative feedback loop where Cas9 cut both the target genomic locus and the AAV vector, thus self-limiting the expression of Cas9. We demonstrated that this system could reduce ∼60% expression of SaCas9 protein and had a 20-fold reduction in off-target activity at 24 weeks post-vector administration in vivo. Moreover, the on-target editing efficacy was not compromised and resulted in a stable reduction in circulating PCSK9 and serum cholesterol. The inclusion of this self-cleavage system in gene-editing approaches could increase the safety profile of AAV-delivered genome-editing nucleases and thereby promote its clinical transformation.http://www.sciencedirect.com/science/article/pii/S2329050121000206AAVCRISPR-Cas9PCSK9self-cleavage |
| spellingShingle | Qian Li Jing Su Yi Liu Xiu Jin Xiaomei Zhong Li Mo Qingnan Wang Hongxin Deng Yang Yang In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system Molecular Therapy: Methods & Clinical Development AAV CRISPR-Cas9 PCSK9 self-cleavage |
| title | In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system |
| title_full | In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system |
| title_fullStr | In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system |
| title_full_unstemmed | In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system |
| title_short | In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system |
| title_sort | in vivo pcsk9 gene editing using an all in one self cleavage aav crispr system |
| topic | AAV CRISPR-Cas9 PCSK9 self-cleavage |
| url | http://www.sciencedirect.com/science/article/pii/S2329050121000206 |
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