Analytical chemistryDec 31, 2025

Light-Activated CRISPR-RNA Using Split Repeats for Simple Cas12a DNA and RNA Detection

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity in detecting clinical samples.

A modular light-activated CRISPR-RNA design was developed using split direct repeat components.
The system allows for easy redesign of the 3' SDR-Spacer to target new sequences while maintaining a universal 5' SDR.
Double-stranded extensions were introduced at the cleavage site to ensure the integrity of the CRISPR function.
This novel approach provides a universal strategy for regulating CRISPR activity, potentially enhancing diagnostic capabilities.
The findings may facilitate broader applications in molecular diagnostics and gene-editing research.

Simplified

Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied todetection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research. Mycoplasma pneumoniae

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Light-Activated CRISPR-RNA Using Split Repeats for Simple Cas12a DNA and RNA Detection

Abstract

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