| Autor: |
Shigemori H; 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, Osaka 565-0871, Japan.; Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada-ku, Kobe, Hyogo 657-0011, Japan., Fujita S; 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, Osaka 565-0871, Japan., Tamiya E; 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, Osaka 565-0871, Japan.; Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan., Wakida SI; 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, Osaka 565-0871, Japan.; Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan., Nagai H; 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, Osaka 565-0871, Japan.; Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada-ku, Kobe, Hyogo 657-0011, Japan. |
| Abstrakt: |
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12 (Cas12) system is attracting interest for its potential as a next-generation nucleic acid detection tool. The system can recognize double-stranded DNA (dsDNA) based on Cas12-CRISPR RNA (crRNA) and induce signal transduction by collateral cleavage. This property is expected to simplify comprehensive genotyping. Here, we report a solid-phase collateral cleavage (SPCC) reaction by CRISPR/Cas12 and its application toward one-pot multiplex dsDNA detection with minimal operational steps. In the sensor, Cas12-crRNA and single-stranded DNA (ssDNA) are immobilized on the sensing surface and act as enzyme and reporter substrates, respectively. We also report a dual-target dsDNA sensor prepared by immobilizing Cas12-crRNA and a fluorophore-labeled ssDNA reporter on separate spots. When a spot captures a target dsDNA sequence, it cleaves the ssDNA reporter on the same spot and reduces its fluorescence by 42.1-57.3%. Crucially, spots targeting different sequences do not show a reduction in fluorescence, thus confirming the one-pot multiplex dsDNA detection by SPCC. Furthermore, the sequence specificity has a two-base resolution, and the detectable concentration for the target dsDNA is at least 10 -9 M. In the future, the SPCC-based sensor array could achieve one-pot comprehensive genotyping by using an array spotter as a reagent-immobilizing method. |
