Versatile and iterative prokaryotic genome engineering through CRISPR-Transposon phage particles

The engineering of microbial genomes is key for the generation of microbial chassis tailored towards human innovation and biotechnological applications. However, prokaryotic genome engineering has been greatly hindered by the innate feasibility, scales, and ease of delivery and integration of genetic payloads in non-model microbial organisms. Here we address these challenges through a Phage Assisted CAST Integration (PAINT) system, and its iterative counterpart (iPAINT), which synergistically couples phage packaging and CAST transposition machinery to achieve reliable, streamlined, and highly efficient targeted genome insertions in a wide array of microbial species.

微生物基因组工程是构建适配人类创新需求与生物技术应用的定制化微生物底盘的核心所在。然而，针对非模式微生物开展原核生物基因组工程时，遗传负载的递送与整合在可行性、操作规模及便捷性层面存在的固有瓶颈，极大制约了该领域的发展。本研究借助噬菌体辅助CAST整合（Phage Assisted CAST Integration, PAINT）系统及其迭代版本iPAINT解决了上述挑战：该系统通过协同耦合噬菌体包装与CAST转座装置，可在广泛的微生物物种中实现可靠、流程简化且高效的靶向基因组插入操作。