Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics

Jul 21, 2025Analytical chemistry

Using crRNA to Control CRISPR-Cas12a for Building Sensitive Molecular Diagnostic Circuits

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Abstract

The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 minutes.

An engineered split CRISPR RNA variant restores full activity of the Cas12a nuclease autonomously.
The CREST system integrates biochemical circuits to facilitate on-demand synthesis and reassembly of the split RNA.
This approach addresses challenges in CRISPR diagnostics, including low efficiency in one-pot assays and limited reagent stability.
CREST demonstrates potential for identifying bacterial infections in patients.

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The clustered regularly interspaced short palindromic repeat (CRISPR) system represents a fundamental tool for biotechnological applications. However, the ability to engineer intrinsic components of the CRISPR system to regulate its activity remains limited. Here, we present a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant autonomously restores the full activity of Cas12a nuclease without the need for chemical modifications or exogenous activation stimuli. Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited by Split en-crRNA Transcription) for molecular diagnostics. CREST integrates biochemical circuits that ingeniously harness isothermal amplification to trigger the on-demand synthesis and reassembly of a universal split en-crRNA, addressing longstanding challenges in CRISPR diagnostics, including the low efficiency of one-pot assays, protospacer adjacent motif restriction, the need for crRNA reoptimization, and limited reagent stability. The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability for identifying bacterial infections in patients. This study provides new insights into Cas12a regulation, which is expected to advance the development of controllable CRISPR technologies for diagnostics as well as broader applications such as genome editing and programmable gene expression.

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Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics

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