Baron, Y.Y.BaronSens, J.J.SensLange, L.L.LangeNassauer, L.L.NassauerKlatt, D.D.KlattHoffmann, D.D.HoffmannKleppa, M.J.M.J.KleppaVollmer Barbosa, PhilippePhilippeVollmer BarbosaKeisker, M.M.KeiskerSteinberg, V.V.SteinbergSuerth, J.D.J.D.SuerthVondran, F.W.R.F.W.R.VondranMeyer, J.J.MeyerMorgan, M.M.MorganSchambach, A.A.SchambachGalla, M.M.Galla2022-12-142022-12-142022https://publica.fraunhofer.de/handle/publica/43001110.1016/j.omtn.2021.12.0332-s2.0-85123679429DNA-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.enalpharetroviral vectorCRISPR-Cas9Gag.MS2 chimeragene editinghomology directed repairmultiplexingtransient expressionvirus-like particlesjournal article