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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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.
Lu, Y., Aimetti, A. A., Langer, R. & Gu, Z. Bioresponsive materials. Nat. Rev. Mater 2, 16075 (2017).
Li, J. Y. & Mooney, D. J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater 1, 16071 (2016).
Kanamala, M., Wilson, W. R., Yang, M. M., Palmer, B. D. & Wu, Z. M. Mechanisms and biomaterials in pH-responsive tumour targeted drug delivery: a review. Biomaterials 85, 152–167 (2016).
Wilson, D. S. et al. Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines. Nat. Mater. 9, 923–928 (2010).
Place, E. S., Evans, N. D. & Stevens, M. M. Complexity in biomaterials for tissue engineering. Nat. Mater. 8, 457–470 (2009).
Mura, S., Nicolas, J. & Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 12, 991–1003 (2013).
Baker, S., Thomson, N., Weill, F. X. & Holt, K. E. Genomic insights into the emergence and spread of antimicrobial-resistant bacterial pathogens. Science 360, 733–738 (2018).
Ji, J. F. et al. MicroRNA expression, survival, and response to interferon in liver cancer. N. Engl. J. Med 361, 1437–1447 (2009).
Pardee, K. et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266 (2016).
Takahashi, M. K. et al. A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nat. Commun. 9, 3347 (2018).
Gootenberg, J. S. et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439–444 (2018).
Na, W., Nam, D., Lee, H. & Shin, S. Rapid molecular diagnosis of infectious viruses in microfluidics using DNA hydrogel formation. Biosens. Bioelectron 108, 9–13 (2018).
Venkatesh, S., Wower, J. & Byrne, M. E. Nucleic acid therapeutic carriers with on-demand triggered release. Bioconjugate Chem. 20, 1773–1782 (2009).
Li, J. et al. Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications. Chem. Soc. Rev. 45, 1410–1431 (2016).
English, M. A. et al. Programmable CRISPR-responsive smart materials. Science 365, 780–785 (2019).
Knott, G. J. & Doudna, J. A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
Jiang, W. Y. & Marraffini, L. A. CRISPR-Cas: new tools for genetic manipulations from bacterial immunity systems. Annu. Rev.Microbiol. 69, 209–228 (2015).
Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).
Myhrvold, C. et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science 360, 444–448 (2018).
Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O. & Zhang, F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat. Protoc. 14, 2986–3012 (2019).
Li, S. Y. et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 4, 20 (2018).
Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).
Li, S. Y. et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 28, 491–493 (2018).
Singh, D. et al. Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc. Natl. Acad. Sci. USA 115, 5444–5449 (2018).
Strohkendl, I., Saifuddin, F. A., Rybarski, J. R., Finkelstein, I. J. & Russell, R. Kinetic basis for DNA target specificity of CRISPR-Cas12a. Mol. Cell 71, 816–824 (2018).
Carlson, R. The changing economics of DNA synthesis. Nat. Biotechnol. 27, 1091–1094 (2009).
Gao, M., Gawel, K. & Stokke, B. T. Toehold of dsDNA exchange affects the hydrogel swelling kinetics of a polymer-dsDNA hybrid hydrogel. Soft Matter 7, 1741–1746 (2011).
Sicilia, G. et al. Programmable polymer-DNA hydrogels with dual input and multiscale responses. Biomater. Sci. 2, 203–211 (2014).
Fern, J. & Schulman, R. Modular DNA strand-displacement controllers for directing material expansion. Nat. Commun 9, 3766 (2018).
Cangialosi, A. et al. DNA sequence-directed shape change of photopatterned hydrogels via high-degree swelling. Science 357, 1126–1129 (2017).
Hajian, R. et al. Detection of unamplified target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor. Nat. Biomed. Eng 3, 427–437 (2019).
Teng, F. et al. Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds. Genome Biol. 20, 15 (2019).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Hu, W. K., Wang, Z. J., Xiao, Y., Zhang, S. M. & Wang, J. L. Advances in crosslinking strategies of biomedical hydrogels. Biomater. Sci. 7, 843–855 (2019).
Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).
Barker, K. et al. Biodegradable DNA-enabled poly(ethylene glycol) hydrogels prepared by copper-free click chemistry. J. Biomater. Sci. Polym. Ed. 27, 22–39 (2016).
Phelps, E. A. et al. Maleimide cross-linked bioactive PEG hydrogel exhibits improved reaction kinetics and cross-linking for cell encapsulation and in situ delivery. Adv. Mater. 24, 64–70 (2012).
Harrington, L. B. et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839–842 (2018).
Caliari, S. R. & Burdick, J. A. A practical guide to hydrogels for cell culture. Nat. Methods 13, 405–414 (2016).
Gobaa, S., Gayet, R. V. & Lutolf, M. P. Artificial niche microarrays for identifying extrinsic cell-fate determinants. Methods Cell Biol. 148, 51–69 (2018).
Previtera, M. L. & Langrana, N. A. Preparation of DNA-crosslinked polyacrylamide hydrogels. J. Vis. Exp. 90, e51323 (2014).
Turkevich, J., Stevenson, P. C. & Hillier, J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc 11, 55–75 (1951).
Wei, X. F. et al. Target-responsive DNA hydrogel mediated “stop-flow” microfluidic paper-based analytic device for rapid, portable and visual detection of multiple targets. Anal. Chem. 87, 4275–4282 (2015).
Masters, J. R. & Stacey, G. N. Changing medium and passaging cell lines. Nat. Protoc. 2, 2276–2284 (2007).
Lee, H. H., Chou, K. S. & Huang, K. C. Inkjet printing of nanosized silver colloids. Nanotechnology 16, 2436–2441 (2005).
Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).
Lin, L. et al. Engineering the direct repeat sequence of crRNA for optimization of FnCpf1-mediated genome editing in human cells. Mol. Ther. 26, 2650–2657 (2018).
Liu, P. P. et al. Enhanced Cas12a editing in mammalian cells and zebrafish. Nucleic Acids Res. 47, 4169–4180 (2019).
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.
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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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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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