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

Abstract

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.

Keywords: CRISPR/Cas12; accumulated sensor array; collateral cleavage; genotyping; miniaturization; multiplex detection; non-contact bioprinting; printed biosensor; protein array.

Figure A1

dsDNA detection on the SPCC-based sensor array after shaking-based washing: (a) HEX-fluorescence images after the incubation of 0 or 10 nM target dsDNA for two hours, (b) Multi-colored image of HEX- and FAM-fluorescence and expected interfaces of each region.

Figure 1

Graphical image of the miniaturized patterning of the sensor array based on non-contact printing and identification of the target dsDNA sequence by the solid-phase collateral cleavage (SPCC)-derived fluorescence decrease on a certain spot.

Figure 2

Overview of the printing box of the AD1520 aspirate/dispense system and external devices. The printing box was built on the basic unit of the system (AD1520 aspirate/dispense system) and extra units, vacuum pump (VP5000 series) and humidifier (Humidifier Ultrasonic U300). As the extra units, vacuum pump (VP5000 series) was connected to vacuum area of AD1520 aspirate/dispense system via air tube to remove residual droplets on outside of nozzle, and humidifier (Humidifier Ultrasonic U300) was connected to upper-right side of AD1520 aspirate/dispense system via air tube to maintain around 95% of the humidity inside the printing box.

Figure 3

Collateral cleavage activity of surface-immobilized Cas12–crRNA in various immobilization-time conditions. (a) Schematic image of the dispensing/immobilization of the Cas12–crRNA and fluorescence-based collateral cleavage activity test. (b) Relationship between fluorescence intensity and the immobilization time of the Cas12–crRNA.

Figure 4

Collateral cleavage activity of the surface-immobilized Cas12–crRNA dispensed by a non-contact printer or a micropipette. (a) Schematic image of the dispensing/immobilization of Cas12–crRNA and the fluorescence-based collateral cleavage activity test. (b) Fluorescence intensity of the dsDNA/F-Q ssDNA reporter solution incubated on each surface (Student’s t-test result; n.s.: not significant, **: p < 0.005, ***: p < 0.0005).

Figure 5

Optimization of washing times after immobilizing Cas12. (a) Alignment of spot with pVenus-N1-targeting crRNA. (b) Schematic image of HEX- and FAM-labeling. (c) FAM-fluorescence images of each washing condition. (d) Cas12-immobilized area derived from each FAM-fluorescence image. (e) FAM-fluorescence intensity of each condition (Student’s t-test result; n.s.: not significant, *: p < 0.05, *******: p < 0.00000005).

Figure 6

dsDNA concentration response on the non-contact-patterned SPCC-based sensor array. (a) Spot diameter comparison in representative HEX-fluorescence images of each printing volume condition ((Target dsDNA) = 0 nM). (b) HEX-fluorescence image in each printing volume of Cas12–crRNA and dsDNA concentration. (c–e) Calibration plots of the cleavage ratio at yellow circle areas at 40, 20, and 10 nL-printing volumes, respectively.

Figure 7

One-pot triple-target dsDNA detection on the non-contact-patterned SPCC-based sensor array fabricated by the optimized process. (a) HEX-fluorescence image of each spot. (b) Cleavage ratio in each crRNA sequence condition after incubation of each dsDNA sample (Student’s t-test result; n.s.: not significant, *: p < 5 × 10⁻², ***: p < 5 × 10⁻⁶, ****: p < 5 × 10⁻⁸).