Shigemori Hiroki, Fujita Satoshi, Tamiya Eiichi, Nagai Hidenori
Advanced Photonics and Biosensing Open Innovation Laboratory (PhotoBIO-OIL), National Institute of Advanced Industrial Science and Technology (AIST), Photonics Center Osaka University, 2-1 Yamada-Oka, Suita 565-0871, Osaka, Japan.
Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada-ku, Kobe 657-0011, Hyogo, Japan.
Micromachines (Basel). 2024 Jan 17;15(1):144. doi: 10.3390/mi15010144.
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.
DNA微阵列已被应用于全面的基因分型，但在复杂操作方面仍存在缺陷。作为一种解决方案，我们之前报道了基于成簇规律间隔短回文重复序列/CRISPR相关蛋白12（CRISPR/Cas12）的固相旁侧切割（SPCC）系统。表面固定的Cas12 - CRISPR RNA（crRNA）可直接与目标双链DNA（dsDNA）杂交，随后通过切割固定在同一位点的单链DNA（ssDNA）报告分子产生信号。因此，基于SPCC的多重dsDNA检测可以轻松进行。本研究报告了通过非接触式打印机对基于SPCC的斑点进行小型化及其在大量积累阵列上进行全面基因分型的性能。最初，建立了Cas12 - crRNA的打印、固定和洗涤过程，以制造基于非接触式图案化SPCC的传感器阵列。基于所开发的传感器阵列获得了目标dsDNA浓度响应，即使斑点直径为0.64±0.05毫米。此外，Cas12 - crRNA的打印量为40、20和10纳升时，检测限分别为572皮摩尔、531皮摩尔和3.04纳摩尔。此外，该传感器阵列在一锅多重检测中特异性地检测了三个dsDNA序列；因此，证实了全面基因分型的可行性。这些结果表明，我们的技术可以通过使用非接触式打印小型化为基于CRISPR/Cas12的微阵列。未来，基于非接触式图案化SPCC的传感器阵列可作为DNA微阵列的替代工具应用。
