Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12...

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Bibliographic Details
Published in:Micromachines (Basel) 2024-01, Vol.15 (1), p.144
Main Authors: Shigemori, Hiroki, Fujita, Satoshi, Tamiya, Eiichi, Nagai, Hidenori
Format: Article
Language:English
Subjects:
Cleavage
collateral cleavage
CRISPR
CRISPR/Cas12
DNA
DNA chips
DNA microarrays
Drug resistance
Gene amplification
Genomics
genotyping
Miniaturization
multiplex detection
Multiplexing
Mutation
Polymerase chain reaction
printed biosensor
Printing
protein array
Reagents
RNA
Sensor arrays
Sensors
Sepsis
Solid phases
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Summary:DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.
ISSN:2072-666X
2072-666X
DOI:10.3390/mi15010144