Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene
The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. T...
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| Format: | Article |
| Language: | English |
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Elsevier
2020-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050120301856 |
| _version_ | 1818435675363475456 |
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| author | Jon R. Backstrom Jinsong Sheng Michael C. Wang Alexandra Bernardo-Colón Tonia S. Rex |
| author_facet | Jon R. Backstrom Jinsong Sheng Michael C. Wang Alexandra Bernardo-Colón Tonia S. Rex |
| author_sort | Jon R. Backstrom |
| collection | DOAJ |
| description | The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo. First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro. Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 (Pcsk9) mRNA and secreted protein 5-fold in vitro. The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo. This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo. |
| first_indexed | 2024-12-14T16:56:39Z |
| format | Article |
| id | doaj.art-e62d5d2d855a481eac4ae831ee2cf1cb |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-14T16:56:39Z |
| publishDate | 2020-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-e62d5d2d855a481eac4ae831ee2cf1cb2022-12-21T22:53:57ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-12-0119139148Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous GeneJon R. Backstrom0Jinsong Sheng1Michael C. Wang2Alexandra Bernardo-Colón3Tonia S. Rex4Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USAVanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USADepartment of Ophthalmology & Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232, USAVanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USAVanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Ophthalmology & Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Corresponding author: Tonia S. Rex, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA.The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo. First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro. Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 (Pcsk9) mRNA and secreted protein 5-fold in vitro. The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo. This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo.http://www.sciencedirect.com/science/article/pii/S2329050120301856CRISPRideactivated Cas9adeno-associated viruscardiovascular diseasein vivoKRAB |
| spellingShingle | Jon R. Backstrom Jinsong Sheng Michael C. Wang Alexandra Bernardo-Colón Tonia S. Rex Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene Molecular Therapy: Methods & Clinical Development CRISPRi deactivated Cas9 adeno-associated virus cardiovascular disease in vivo KRAB |
| title | Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene |
| title_full | Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene |
| title_fullStr | Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene |
| title_full_unstemmed | Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene |
| title_short | Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene |
| title_sort | optimization of s aureus dcas9 and crispri elements for a single adeno associated virus that targets an endogenous gene |
| topic | CRISPRi deactivated Cas9 adeno-associated virus cardiovascular disease in vivo KRAB |
| url | http://www.sciencedirect.com/science/article/pii/S2329050120301856 |
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