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This study addresses a novel formulation method, focusing on organic solvent\u2010free lipid nanoparticles for the efficient delivery of CRISPR\u2010Cas9 components. Therefore, novel stealth lipids comprising the lipid anchor cholesterol hemisuccinate conjugated to poly(2\u2010ethyl\u20102\u2010oxazoline) (PEtOx) were synthesized and evaluated as a promising in vitro alternative to commercially available poly(ethylene glycol) (PEG)\u2010based lipids. Our study identified PEtOx\u2010lipids as markedly more biocompatible compared to PEG\u2010based analogues (12\u2010fold reduction in cytotoxicity; 99% reduced hemolytic effect) with high efficiency for mRNA gene delivery. We demonstrate successful CRISPR\u2010Cas9 activity ex vivo and efficient uptake of PEtOx nanoparticles by macrophages in vivo (66.5% pos. F4\/80\n                    <jats:sup>+<\/jats:sup>\n                    macrophages). Our data demonstrate that PEtOx\u2010based stealth lipids are a promising alternative to PEG. 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