CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.

中文翻译：

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用于基于 CRISPR 的哺乳动物基因组编辑的工程杆状病毒蛋白和 DNA 共传递系统

基于 CRISPR 的 DNA 编辑技术使真核细胞的基因组工程能够快速且易于实现。然而，遗传编码的 CRISPR 组件的传递仍然具有挑战性，持续的 Cas9 表达与较高的脱靶活性相关，可以通过 Cas9 蛋白传递来减少脱靶活性。在这里，我们证明杆状病毒及其 DNA 货物可用于包装蛋白质并将其递送至人类细胞。使用蛋白质负载杆状病毒 (pBV)，我们展示了 Cas9 或碱基编辑器蛋白质的传递，从而在人类细胞中实现有效的基因组和碱基编辑。通过实施可逆的化学诱导异二聚化系统，我们表明蛋白质货物可以选择性且更有效地加载到 pBV (spBV) 中。使用 spBV，我们在一组人类细胞系中实现了高水平的多重基因组编辑。重要的是，spBV 在没有可检测到的脱靶事件的情况下仍保持高编辑效率。最后，通过利用 Cas9 蛋白和模板 DNA 共传递，我们在一组人类细胞系中使用单个 spBV 证明了 1.8 kb 异源 DNA 负载的高达 5% 的位点特异性靶向整合。总之，我们证明 spBV 代表了需要共同递送 DNA 和蛋白质货物的 CRISPR 应用的一种多功能、高效且可能更安全的替代方案。