Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube

Dec 6, 2025Analytica chimica acta

Using a CRISPR-Cas12a system with split guides to detect DNA and RNA one after the other in a single tube

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Abstract

The limit of detection was 0.70 pM for HBV DNA and 0.47 nM for HIV RNA.

CRISPR Cas12a with split crRNA can detect both HBV DNA and HIV RNA in a single tube.
Initial detection of HBV DNA triggers fluorescence changes correlated with its concentration after 1 hour.
The method allows for subsequent quantification of HIV RNA without interference from the previous step.
This approach simplifies the detection process and requires only Cas12a protein in one tube.
The strategy has potential for semi-quantifying two types of DNA simultaneously.

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Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.

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Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube

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