A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics

Oct 30, 2025Analytical chemistry

A Light-Controlled CRISPR System for Fast and Universal Disease Detection

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Abstract

The developed CRISPR system achieved 95.7% sensitivity and 100% specificity in detecting human papillomavirus 16 DNA in 50 clinical samples.

A photo-controllable CRISPR RNA (crRNA) was created by linking it to a G-quadruplex block.
Irradiation with light removes the G-quadruplex, enabling the crRNA to hybridize with the DNA target.
This method demonstrated detection efficiency comparable to qPCR but was faster and more cost-effective.
The approach could simplify diagnostic processes through the use of lateral flow test strips.

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The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.

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A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics

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