DNA Origami‐Based Single‐Molecule CRISPR Machines for Spatially Resolved Searching

Abstract Repurposing the RNA‐guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR‐Cas9‐based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami‐based single‐molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially‐confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high‐throughput nucleic acid detection.