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

Molecular Therapy: Nucleic Acids (Dec 2019)

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

Affiliations

Fabian Lindel: 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
Carolin R. Dodt: 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
Niklas Weidner: 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
Monique Noll: 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
Fabian Bergemann: 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
Rayk Behrendt: Institute of Immunology, Technische Universität Dresden, 01307 Dresden, Germany
Sarah Fischer: 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
Josephine Dietrich: Cellex Patient Treatment GmbH, 01307 Dresden, Germany
Marc Cartellieri: Cellex Patient Treatment GmbH, 01307 Dresden, Germany
Martin V. Hamann: 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
Dirk Lindemann: 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; Corresponding author: Dirk Lindemann, Institute of Virology, Medical Faculty “Carl Gustav Carus,” Technische Universität Dresden, 01307 Dresden, Germany.

Journal volume & issue: Vol. 18
pp. 708 – 726

Abstract

Read online

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

Published in Molecular Therapy: Nucleic Acids

ISSN: 2162-2531 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Medicine: Therapeutics. Pharmacology
Website: https://www.cell.com/molecular-therapy-family/nucleic-acids/latest-content

About the journal