Lindel Fabian, Dodt Carolin R, Weidner Niklas, Noll Monique, Bergemann Fabian, Behrendt Rayk, Fischer Sarah, Dietrich Josephine, Cartellieri Marc, Hamann Martin V, Lindemann Dirk
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.
Institute of Immunology, Technische Universität Dresden, 01307 Dresden, Germany.
Mol Ther Nucleic Acids. 2019 Dec 6;18:708-726. doi: 10.1016/j.omtn.2019.10.006. Epub 2019 Oct 17.
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.
将CRISPR/Cas技术应用于哺乳动物已彻底改变了基因组工程。特别是在临床应用方面，非常希望在狭窄的时间范围内高效表达Cas9，以尽量减少脱靶编辑的积累。我们开发了一种有效的、不依赖适体的逆转录病毒递送系统，用于递送Cas9 mRNA，该系统利用了泡沫病毒（FV）的一种独特能力：高效包装和转移非病毒RNA。这使我们能够创建一个FV载体工具箱，用于将CRISPR/Cas9组件高效、瞬时递送至不同靶组织（TraFo）。与CRISPR/Cas9慢病毒载体系统相比，通过TraFo-Cas9载体共递送Cas9 mRNA并结合逆转录病毒、整合缺陷型单向导RNA（sgRNA）表达，增强了基因失活的效力和特异性。此外，单独的TraFo-Cas9递送允许选择性地加入修复基质以进行有效的基因校正或标记，以及加入荧光阴性选择标记以易于识别脱靶编辑或错误修复事件。因此，TraFo CRISPR工具箱代表了一种用于基因失活和基因编辑的有趣替代技术。
