Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release
Author(s)
Gayet, Raphael V.
de Puig Guixe, Helena
English, Max A.
Soenksen Martinez, Luis Ruben
Nguyen, Peter Q.
Mao, Angelo S.
Angenent-Mari, Nicolaas M.
Collins, James J.
Date Issued
August 2020
Journal
Nature Protocols
Publisher
Springer Science and Business Media LLC
Citation
Gayet, Raphael V. et al. "Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release." Nature Protocols 15, 9 (August 2020): 3030–3063. © 2020 The Author(s)
Version
Author's final manuscript
Abstract
Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid–responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3–4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2–3 d each.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Massachusetts Institute of Technology. Department of Mechanical Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
Terms of Use
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Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1038/s41596-020-0367-8
