The comprehensive detection of nucleic acid in pathogen is crucial to achieving effective prevention of infectious diseases. However, conventional methods (DNA sequencing etc.) take a long time (2~24 h) for the detection of the target sequences. Moreover, the accuracy of the sequence readout is important to provide the proper treatment to patients.
Recently, CRISPR/Cas12-based DNA recognition is paid attention for rapid (<1 h) and high specific recognition to target sequence. According to CRISPR/Cas12-based diagnostics, the target double-strand DNA (dsDNA) sequence is recognized by the complex of Cas12 and guide RNA (gRNA) with a sequence complementary to the target. Subsequently, the Cas12 generates the signal (fluorescence etc.) by using single-strand deoxyribonuclease activation. While CRISPR/Cas12-based diagnostic have many advantages, the above-mentioned cleavage makes it difficult for multiplex detection in a single reaction chamber.
To achieve the one-pot comprehensive dsDNA detection by CRISPR/Cas12, we have been developing a DNA sensor array by integrating the sensing spots immobilized with both Cas12-gRNA and fluorophore-labeled ssDNA reporter. When the target dsDNA is applied on the array, it is captured by the Cas12 spot with complementary gRNA sequence. At the spot, ssDNA reporter immobilized adjacent to the activated Cas12 is cleaved by and the target dsDNA is identified as the spot with low fluorescence.
Firstly, we fabricated the CRISPR/Cas12 sensor array and tried to identify two target dsDNA sequences between EGFP and its two-base mutation (MUT). When the spot captured specific target, the fluorescence was decreased 6.0~17.2-fold compared to that of the other spot (Fig. 2). Furthermore, it was appeared that 100 nM target dsDNA can be detected in 20 min by the spot.
Overall, CRISPR/Cas12-immobilized surface was confirmed to detect two target dsDNA with short reaction time. For future prospects, we will attempt to simultaneously detect ≥1000 targets by accumulating the spots using a droplet dispenser.

Overview of this study

Fluorescence of two-plex dsDNA detection

Eiichi Tamiya

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