Cell extract-based in vitro DNA replication enables sustainable cell-free gene expression

Cell extract-based cell-free gene expression (CFE) systems break cellular boundaries and create emerging platforms for synthetic biology applications. However, these systems lack intrinsic DNA replication capability, thereby limiting sustained transcription and translation in a cell-free environment. Here, we introduce a LoopReX system based on E. coli CFE by integration of the DNA replication machinery, enabling iterative in vitro DNA replication and sustainable cell-free gene expression. We establish LoopReX by integrating phi29 DNA polymerase into the crude lysate-based E. coli CFE system, allowing for coupled DNA replication and protein expression. We elucidate the synergetic mechanisms between T7 RNA polymerase and phi29 DNA polymerase in facilitating in vitro DNA replication. Additionally, we optimize LoopReX reaction conditions by machine learning strategies. Finally, we demonstrate that the enhanced LoopReX system achieves highly efficient DNA replication and protein production in an iterative, scalable, and sustainable manner. We anticipate that LoopReX will significantly advance the application of CFE in synthetic biology.