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A Triple-Mismatch Differentiating assay exploiting activation and trans cleavage of CRISPR-Cas12a for mutation detection with ultra specificity and sensitivity. | LitMetric

AI Article Synopsis

  • Liquid biopsy technology is a non-invasive method for cancer screening, with CRISPR-Cas12a-based detection showing high sensitivity and ease of use.
  • The TMD (Triple-Mismatch Differentiating) assay enhances CRISPR-Cas12a's ability to distinguish between single-base mismatches by amplifying minor thermodynamic differences at multiple sites.
  • Testing with pancreatic cancer samples indicated that TMD assay results closely matched next-generation sequencing (NGS) outcomes, suggesting its potential for early cancer detection in clinical settings.

Article Abstract

Liquid biopsy technology is non-invasive and convenient, and is currently an emerging technology for cancer screening. Among them, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12a (Cas12a) based nucleic acid detection technology has the advantages of high sensitivity, rapidity, and easy operation. However, CRISPR-Cas12a does not discriminate single-base mismatches of targets well enough to meet the needs of clinical detection. Herein, we developed the Triple-Mismatch Differentiating (TMD) assay. This assay amplified the small thermodynamic difference in mismatches at one site at the level of CRISPR-Cas12a activation to a significant thermodynamic difference at three sites at both the level of CRISPR-Cas12a activation and trans-cleavage, which greatly improves the ability of CRISPR-Cas12a to discriminate between base mismatches. Our manipulation greatly improved the specificity of the CRISPR-Cas12a system while maintaining its inherent sensitivity and simplicity, increasing the detection limit to 0.0001%. When testing samples from pancreatic cancer patients, our results were highly consistent with NGS sequencing results. We believe that the TMD assay will provide a new technology for early cancer detection and will be widely used in the clinical practice.

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Source
http://dx.doi.org/10.1016/j.bios.2024.116826DOI Listing

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