Photocontrolled Programmable Enzymatic Cascade for Robust CRISPR Diagnostics

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.