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

摘要

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.