Foldback-crRNA-Enhanced CRISPR/Cas13a System (FCECas13a) Enables Direct Detection of Ultrashort sncRNA

Anal Chem. 2023 Oct 24;95(42):15606-15613. doi: 10.1021/acs.analchem.3c02687. Epub 2023 Oct 12.

Abstract

The CRISPR/Cas13a system has promising applications in clinical small noncoding RNA (sncRNA) detection because it is free from the interference of genomic DNA. However, detecting ultrashort sncRNAs (less than 20 nucleotides) has been challenging because the Cas13a nuclease requires longer crRNA-target RNA hybrids to be activated. Here, we report the development of a foldback-crRNA-enhanced CRISPR/Cas13a (FCECas13a) system that overcomes the limitations of the current CRISPR/Cas13a system in detecting ultrashort sncRNAs. The FCECas13a system employs a 3'-terminal foldback crRNA that hybridizes with the target ultrashort sncRNA, forming a double strand that "tricks" the Cas13a nuclease into activating the HEPN structural domain and generating trans-cleavage activity. The FCECas13a system can accurately detect miRNA720 (a sncRNA currently known as tRNA-derived small RNA), which is only 17 nucleotides long and has a concentration as low as 15 fM within 20 min. This FCECas13a system opens new avenues for ultrashort sncRNA detection with significant implications for basic biological research, disease prognosis, and molecular diagnosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / metabolism
  • Nucleotides
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Small Untranslated*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Small Untranslated
  • Endonucleases
  • Nucleotides