Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of proteins and RNAs, including gene editing agents. We report a system for the laboratory evolution of eVLPs that enables the discovery of eVLP variants with improved properties. The system uses barcoded guide RNAs loaded within DNA-free eVLP-packaged cargos to uniquely label each eVLP variant in a library, enabling the identification of desired variants following selections for desired properties. We applied this system to mutate and select eVLP capsids with improved eVLP production properties or transduction efficiencies in human cells. By combining beneficial capsid mutations, we developed fifth-generation (v5) eVLPs, which exhibit a 2–4-fold increase in cultured mammalian cell delivery potency compared to previous-best v4 eVLPs. Analyses of v5 eVLPs suggest that these capsid mutations optimize packaging and delivery of desired ribonucleoprotein cargos rather than native viral genomes and substantially alter eVLP capsid structure. These findings suggest the potential of barcoded eVLP evolution to support the development of improved eVLPs.

工程病毒样颗粒 （eVLP） 是蛋白质和 RNA（包括基因编辑剂）瞬时递送的有前途的载体。我们报道了一种用于 eVLP 实验室进化的系统，该系统能够发现具有改进特性的 eVLP 变体。该系统使用载入无 DNA 的 eVLP 包装货物中的条形码向导 RNA 来唯一标记文库中的每个 eVLP 变体，从而能够在选择所需特性后识别所需的变体。我们应用该系统来突变和选择在人类细胞中具有改进的 eVLP 生产特性或转导效率的 eVLP 衣壳。通过结合有益的衣壳突变，我们开发了第五代 （v5） eVLP，与以前最好的 v4 eVLP 相比，培养的哺乳动物细胞递送效力提高了 2-4 倍。对 v5 eVLP 的分析表明，这些衣壳突变优化了所需核糖核蛋白货物的包装和递送，而不是天然病毒基因组，并显着改变了 eVLP 衣壳结构。这些发现表明条形码 eVLP 进化的潜力可以支持改进的 eVLP 的开发。