TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components
Research output: Contribution to journal › Research article › Contributed › peer-review
Contributors
Abstract
The adaptation of CRISPR/Cas technology for use in mammals has revolutionized genome engineering. In particular with regard to clinical application, efficient expression of Cas9 within a narrow time frame is highly desirable to minimize the accumulation of off-target editing. We developed an effective, aptamer-independent retroviral delivery system for Cas9 mRNAs that takes advantage of a unique foamy virus (FV) capability: the efficient encapsidation and transfer of non-viral RNAs. This enabled us to create a FV vector toolbox for efficient, transient delivery (TraFo) of CRISPR/Cas9 components into different target tissues. Co-delivery of Cas9 mRNA by TraFo-Cas9 vectors in combination with retroviral, integration-deficient single guide RNA (sgRNA) expression enhanced efficacy and specificity of gene-inactivation compared with CRISPR/Cas9 lentiviral vector systems. Furthermore, separate TraFo-Cas9 delivery allowed the optional inclusion of a repair matrix for efficient gene correction or tagging as well as the addition of fluorescent negative selection markers for easy identification of off-target editing or incorrect repair events. Thus, the TraFo CRISPR toolbox represents an interesting alternative technology for gene inactivation and gene editing.
Details
Original language | English |
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Pages (from-to) | 708-726 |
Number of pages | 19 |
Journal | Molecular therapy. Nucleic acids |
Volume | 18 |
Publication status | Published - 6 Dec 2019 |
Peer-reviewed | Yes |
External IDs
PubMedCentral | PMC6859288 |
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Scopus | 85074671012 |
ORCID | /0000-0002-0320-4223/work/150884956 |