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Related Concept Videos

CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Hybrid Printing for the Fabrication of Smart Sensors
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Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing.

Hiroki Shigemori1,2, Satoshi Fujita1, Eiichi Tamiya1,3

  • 1Advanced 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.

Micromachines
|January 23, 2024
PubMed
Summary

This study miniaturizes the CRISPR/Cas12-based solid-phase collateral cleavage (SPCC) system using non-contact printing for easier comprehensive genotyping. The developed sensor array enables multiplexed DNA detection, offering a potential alternative to traditional DNA microarrays.

Keywords:
CRISPR/Cas12accumulated sensor arraycollateral cleavagegenotypingminiaturizationmultiplex detectionnon-contact bioprintingprinted biosensorprotein array

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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.