Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA

Anal Chem. 2024 Mar 12;96(10):4322-4329. doi: 10.1021/acs.analchem.4c00313. Epub 2024 Feb 29.

Abstract

As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 103 particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • DNA / genetics
  • Humans
  • MicroRNAs* / genetics
  • MicroRNAs* / metabolism
  • Neoplasms* / diagnosis
  • Neoplasms* / genetics
  • RNA, Guide, CRISPR-Cas Systems

Substances

  • MicroRNAs
  • RNA, Guide, CRISPR-Cas Systems
  • DNA