High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA

Anal Chem. 2021 Feb 2;93(4):2038-2044. doi: 10.1021/acs.analchem.0c03708. Epub 2021 Jan 7.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosensing Techniques
  • CRISPR-Cas Systems*
  • Cell Line
  • DNA, Catalytic / genetics
  • DNA, Catalytic / metabolism*
  • Humans
  • MicroRNAs / chemistry*
  • Protein Conformation

Substances

  • DNA, Catalytic
  • MicroRNAs