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

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• DNA microarrays are essential for comprehensive genotyping but involve complex procedures.
• The solid-phase collateral cleavage (SPCC) system using CRISPR/Cas12 offers a simpler approach for DNA detection.
• Previous work established SPCC for multiplexed double-stranded DNA (dsDNA) detection.

Purpose of the Study:

• To miniaturize the SPCC system using non-contact printing for enhanced DNA microarray applications.
• To evaluate the performance of the miniaturized SPCC sensor array for comprehensive genotyping.
• To demonstrate the feasibility of multiplexed detection on a massively accumulated array.

Main Methods:

• Fabrication of a non-contact-patterned SPCC sensor array by optimizing printing, immobilization, and washing of Cas12-CRISPR RNA (crRNA).
• Characterization of the sensor array's response to target dsDNA concentration with small spot sizes (0.64 ± 0.05 mm).
• Assessment of the limit of detection (LOD) based on varying printing volumes of Cas12-crRNA.

Main Results:

• Successful fabrication of a miniaturized SPCC sensor array using non-contact printing.
• Demonstrated dsDNA concentration response with high sensitivity, achieving low limits of detection (e.g., 531 pM).
• Confirmed feasibility of one-pot multiplexed detection of three distinct dsDNA sequences for comprehensive genotyping.

Conclusions:

• The non-contact printing approach successfully miniaturizes the CRISPR/Cas12-based SPCC system into a microarray format.
• This technology provides an efficient and simplified method for comprehensive genotyping.
• The developed sensor array presents a promising alternative to conventional DNA microarrays for various genomic applications.