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

Nat Protoc. 2020 Sep;15(9):3030-3063. doi: 10.1038/s41596-020-0367-8. Epub 2020 Aug 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acrylic Resins / chemistry
  • Bacterial Proteins / metabolism
  • Base Sequence
  • CRISPR-Associated Proteins / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA / chemistry
  • DNA / genetics
  • Diagnostic Techniques and Procedures*
  • Drug Delivery Systems / methods*
  • Drug Liberation*
  • Endodeoxyribonucleases / metabolism
  • Humans
  • K562 Cells
  • Polyethylene Glycols / chemistry
  • RNA, Guide / genetics
  • Smart Materials / chemistry*

Substances

  • Acrylic Resins
  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide
  • Smart Materials
  • Polyethylene Glycols
  • polyacrylamide
  • DNA
  • Cas12a protein
  • Endodeoxyribonucleases