DNA Origami Post‐Processing by CRISPR‐Cas12a

Abstract

Customizable nanostructures built through the DNA‐origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting‐edge tools for DNA‐origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA‐origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA‐origami structures. We target single‐stranded (ss) regions of DNA‐origami structures and remove them with CRISPR‐Cas12a, a hyper‐active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post‐processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a‐like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.

DNA origami

“…A DNA origami 'force clamp' was built, where a U-shaped body suspends a segment of DNA under a defined tension of 0-12 pN (fig. 6e) and enables single-molecule force spectroscopy studies of tension-dependent Holliday junction isomerization 240 , DNA nuclease activities 241 and formation of the transcription pre-initiation complex 242 . Barrel or clamp-shaped 3D DNA origami structures have found applications in the cryo-EM structural determination of proteins, where DNA nanostructures serve to define the thickness of a vitrified ice sheet 243 , create a hydrophobic environment to stabilize membrane proteins 244 and orient DNA-binding proteins at desired rotation angles 245 .…”

Strategies for Constructing and Operating DNA Origami Linear Actuators

“…Alternative ways to cut single-stranded links within DNA origami include use of self-cleaving DNAzymes [36] or RNA-guided endonucleases. [37] In our alternative two-pot assembly, we used a standard-size scaffold (p8064) to create the rail and a shorter phagemid-based [38] scaffold (2880 nt) to create the slider. In both strategies, we chose to base the rail on a six-helix-bundle honeycomb design, offering a reasonable compromise between material use and rigidity: previous studies [39][40][41] indicate that this design has a persistence length above 1 µm.…”

Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

“…(B) adapted with permission from ( Wang et al., 2020a ); Published (2020) by The American Association for the Advancement of Science. (C) adapted with permission from ( Xiong et al., 2020 ); Copyright (2019) John Wiley & Sons. (D) adapted with permission from ( Abe et al., 2021 ); Copyright (2021) by Royal Society of Chemistry.…”