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Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release

Nature Protocols volume 15, pages 3030–3063 (2020)Cite this article

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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.

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Fig. 1: Activation of Cas12a collateral ssDNA cleavage activity by trigger dsDNA.
Fig. 2: Design and synthesis of guide RNA molecules (Steps 1–14).
Fig. 3: Synthesis of PEG hydrogels harboring ssDNA-tethered molecules (Step 19A).
Fig. 4: Monitoring the Cas12a-mediated release of fluorophores from PEG hydrogels (Step 19A).
Fig. 5: Synthesis of polyacrylamide hydrogels crosslinked by DNA molecules (Step 19B).
Fig. 6: Monitoring the Cas12a-mediated release of nanoparticles through bulk PA-DNA gel degradation (Step 19C).
Fig. 7: Workflow for the crosslinking and Cas12a-mediated actuation of PA-DNA hydrogels containing encapsulated cells (Step 19D).
Fig. 8: Basal ssDNA cleavage activity of cell culture media.
Fig. 9: Multi-modal detection of DNA and RNA targets using CRISPR-sensitive hydrogels in paper-based fluidic devices.
Fig. 10: Design and assembly of the microfluidic paper-based analytical device (Step 19E(ii–xii)).
Fig. 11: Modification of the paper-fluidic device for analog signal recording.
Fig. 12: Modification of the paper-fluidic device for remote transmission of digital signals.
Fig. 13: Time-dependent drying of PA-DNA hydrogels.
Fig. 14: Anticipated Cas12a activation by dsDNA triggers and subsequent material actuation.

Data availability

The data generated or analyzed during this study are included in this article and our previous publication15. The original data files can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group and the Wyss Institute for Biologically Inspired Engineering, Harvard University (J.J.C., H.d.P., L.R.S., A.S.M.). L.R.S. was also supported by CONACyT grant 342369/408970, and N.M.A.-M. was supported by MIT-TATA Center fellowship 2748460. We thank C. Johnston for helpful comments during manuscript editing.

Author information

Author notes

  1. These authors contributed equally: Raphael V. Gayet, Helena de Puig, Max A. English, Luis R. Soenksen.

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Raphael V. Gayet, Max A. English, Nicolaas M. Angenent-Mari & James J. Collins

  2. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Raphael V. Gayet, Helena de Puig, Max A. English, Luis R. Soenksen, Angelo S. Mao, Nicolaas M. Angenent-Mari & James J. Collins

  3. Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA, USA

    Raphael V. Gayet

  4. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Helena de Puig, Luis R. Soenksen, Peter Q. Nguyen, Angelo S. Mao, Nicolaas M. Angenent-Mari & James J. Collins

  5. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Luis R. Soenksen

  6. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA

    James J. Collins

  7. Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    James J. Collins

  8. Harvard-MIT Program in Health Sciences and Technology, Cambridge, MA, USA

    James J. Collins

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Contributions

R.V.G., H.d.P., M.A.E., L.R.S., P.Q.N., A.S.M. and N.M.A.-M. designed and conducted experiments described in this article and wrote the manuscript. J.J.C. directed the overall research and edited the manuscript.

Corresponding author

Correspondence to James J. Collins.

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Competing interests

R.V.G., H.d.P., M.A.E., L.R.S., P.Q.N., A.S.M., N.M.A.-M. and J.J.C. are inventors on U.S. Patent Application No. 16/778,524, which covers CRISPR-responsive materials. J.J.C. is a co-founder and director of Sherlock Biosciences.

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Peer review information Nature Protocols thanks Chase Beisel, Cole DeForest and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references used in the development of this protocol

English, M. A. et al. Science 365, 780–785 (2019): https://doi.org/10.1126/science.aaw5122

Gootenberg, J. S. et al. Science 356, 438–442 (2017): https://doi.org/10.1126/science.aam9321

Pardee, K. et al. Cell 165, 1255–1266 (2016): https://doi.org/10.1016/j.cell.2016.04.059

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Supplementary Data 1–3.

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Supplementary Software 1

Arduino code to record the RFID signal of the modified µPAD

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Gayet, R.V., de Puig, H., English, M.A. et al. Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release. Nat Protoc 15, 3030–3063 (2020). https://doi.org/10.1038/s41596-020-0367-8

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