TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components

Fabian Lindel; Carolin R Dodt; Niklas Weidner; Monique Noll; Fabian Bergemann; Rayk Behrendt; Sarah Fischer; Josephine Dietrich; Marc Cartellieri; Martin V Hamann; Dirk Lindemann

doi:10.1016/j.omtn.2019.10.006

TraFo-CRISPR: Enhanced Genome Engineering by Transient Foamy Virus Vector-Mediated Delivery of CRISPR/Cas9 Components

Mol Ther Nucleic Acids. 2019 Dec 6:18:708-726. doi: 10.1016/j.omtn.2019.10.006. Epub 2019 Oct 17.

Affiliations

¹ Institute of Virology, Medical Faculty "Carl Gustav Carus," Technische Universität Dresden, 01307 Dresden, Germany; CRTD/DFG-Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany.
² Institute of Immunology, Technische Universität Dresden, 01307 Dresden, Germany.
³ Cellex Patient Treatment GmbH, 01307 Dresden, Germany.
⁴ Institute of Virology, Medical Faculty "Carl Gustav Carus," Technische Universität Dresden, 01307 Dresden, Germany; CRTD/DFG-Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany. Electronic address: dirk.lindemann@tu-dresden.de.

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.

Keywords: CAR T-cell; CRISPR/Cas9; TraFo-Cas9; foamy virus; genome engineering; transient system; viral vector.