Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

• Main Authors: Yvonne Baron (Author), Johanna Sens (Author), Lucas Lange (Author), Larissa Nassauer (Author), Denise Klatt (Author), Dirk Hoffmann (Author), Marc-Jens Kleppa (Author), Philippe Vollmer Barbosa (Author), Maximilian Keisker (Author), Viviane Steinberg (Author), Julia D. Suerth (Author), Florian W.R. Vondran (Author), Johann Meyer (Author), Michael Morgan (Author), Axel Schambach (Author), Melanie Galla (Author)
• Format: Book
• Published: Elsevier, 2022-03-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Item Description

2162-2531
10.1016/j.omtn.2021.12.033