Biosensors & bioelectronicsMay 30, 2026

Detecting pathogens by reading CRISPR signals made from amplified DNA or RNA samples

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated May 31, 2026

Abstract

LAMP-TRAC detected as little as four copies/μL of Mycobacterium tuberculosis genomic DNA within 1 hour.

A noncanonical activation mode of Cas12a was explored to eliminate its dependency on a specific sequence in target DNA.
The method, named PCR followed by Transcription And CRISPR-Cas12a (PCR-TRAC), generates the necessary RNA guide from amplified DNA.
Loop-mediated isothermal amplification (LAMP)-TRAC was developed using this approach to enhance sensitivity and speed.
Both PCR-TRAC and LAMP-TRAC successfully detected Mycobacterium tuberculosis DNA from clinical samples.
LAMP-TRAC demonstrated greater sensitivity and rapid detection compared to PCR-TRAC.

Simplified

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 12a (Cas12a) is an RNA-guided nuclease that has been adapted for nucleic acid-based molecular diagnostics. However, the widespread adoption of Cas12-based molecular diagnostics has been limited by dependency of a protospacer adjacent motif (PAM) sequence within the target double-stranded DNA (dsDNA) required to activate Cas12a. To eliminate PAM dependency, we explored a noncanonical activation mode by designing tailed primers for PCR to generate crRNA from amplified target dsDNA products via transcription. The transcribed crRNA, along with a dsDNA activator, then activates Cas12a. We validated this method and named it PCR followed by Transcription And CRISPR-Cas12a (PCR-TRAC). We then developed loop-mediated isothermal amplification (LAMP)-TRAC using tailed primers. Both methods detected Mycobacterium tuberculosis genomic DNA extracted from clinical samples. LAMP-TRAC was more sensitive and faster than PCR-TRAC, detecting as little as four copies/μL of M. tuberculosis genomic DNA within 1 h. We envision that our CRISPR-Cas12-based diagnostic approach could be expanded to become a universal platform for identifying various other nucleic acid targets.

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Declaration of competing interest The authors declare no competing interests.

PubMed

Detecting pathogens by reading CRISPR signals made from amplified DNA or RNA samples

Abstract

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Funding

Competing interests

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Abstract

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Funding

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Related papers

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