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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Photocontrolled Programmable Enzymatic Cascade for Robust CRISPR Diagnostics.

Menglu Hu1, Yihui Wang1, Weiwei Qi1

  • 1School of Life Sciences, South China Normal University, Guangzhou 510631, China.

Journal of the American Chemical Society
|August 13, 2025
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Summary
This summary is machine-generated.

This study introduces a novel photocontrolled CRISPR diagnostic system for nucleic acid detection. The technology overcomes protospacer adjacent motif limitations and enables simultaneous dual-gene detection, enhancing diagnostic capabilities.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • CRISPR Technology

Background:

  • CRISPR-Cas12a diagnostics offer advanced nucleic acid detection but face limitations.
  • Protospacer adjacent motif (PAM) requirements restrict target site selection.
  • Limited multiplexing capabilities hinder simultaneous detection of multiple targets.

Purpose of the Study:

  • To develop a photocontrolled, one-pot CRISPR diagnostic system.
  • To overcome PAM constraints and enhance multiplexing in CRISPR diagnostics.
  • To enable simultaneous detection of target genes and internal controls for improved clinical utility.

Main Methods:

  • A photocontrolled enzymatic cascade strategy was employed.
  • Sequential reactions included nucleic acid amplification, ssDNA generation via lambda exonuclease, and PAM-independent Cas12a detection.
  • Orthogonal trans-cleavage of Cas12a and Cas13a facilitated dual-gene detection.

Main Results:

  • The system successfully achieved PAM-independent detection.
  • Simultaneous dual-gene detection was demonstrated using Cas12a and Cas13a.
  • Clinical samples of Mycobacterium tuberculosis (MTB) were accurately detected along with an internal control gene (ACTB).

Conclusions:

  • The photocontrolled one-pot CRISPR diagnostic technology enhances flexibility and overcomes limitations of conventional methods.
  • This approach advances the clinical application of CRISPR-based diagnostics by enabling simultaneous target and control gene detection.
  • The developed system shows significant promise for improved molecular diagnostics.