Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection

Angew Chem Int Ed Engl. 2023 Jun 5;62(23):e202300663. doi: 10.1002/anie.202300663. Epub 2023 Apr 27.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

Keywords: CRISPR-Cas12a; Caged crRNA; Nucleic Acid Detection; One-Pot Assay; Photoactivation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agriculture
  • Biological Assay
  • CRISPR-Cas Systems* / genetics
  • Nucleic Acid Amplification Techniques
  • Nucleotidyltransferases
  • RNA, Guide, CRISPR-Cas Systems*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Nucleotidyltransferases