Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

中文翻译：

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杆状病毒载体精确递送人类基因组中的大型DNA货物

通过CRISPR技术进行精确的基因编辑和基因组工程设计，需要同时将多个DNA编码的成分同时递送到活细胞中，从而迅速超过当前使用的病毒载体系统的载重量。在这里，我们利用杆状病毒无与伦比的异源DNA载货能力来解决这一瓶颈。我们实现了混合DNA技术（MultiMate），可在单个杆状病毒载体中快速，无错误地组装当前多达25个功能性DNA模块，从而实现基于CRISPR的基因组工程。利用不依赖同源性的靶向整合（HITI），我们可以在人类细胞中实现高达30％的正确基因组干预，包括将大型DNA有效载荷精确对接在ACTB基因座中。我们展示了杆状病毒载体提供的主要编辑工具包，可实现无缝的DNA搜索和替换干预，从而实现，使用单个载体，即可在HEK3基因座中高效插入无裂解的三核苷酸，而无可检测的插入缺失。因此，我们的方法在人类细胞基因组中释放了广泛的编辑和工程应用。