SDS-CRISPR for Single-Nucleotide Variant Detection

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• CRISPR-Cas12a is a powerful tool for nucleic acid diagnostics but struggles with single-nucleotide variant (SNV) detection due to mismatch tolerance.
• Accurate SNV detection is crucial for applications like cancer variant identification and infectious disease diagnostics.

Purpose of the Study:

• To develop a highly precise CRISPR-Cas12a-based system for allele discrimination and SNV detection.
• To elucidate the structural mechanisms underlying SNV discrimination in the novel system.
• To enable rapid, on-site molecular diagnostics for clinical applications.

Main Methods:

• Development of Structure-Disruption-Sensitive CRISPR (SDS-CRISPR) utilizing AlphaFold3 modeling and bioinformatic analysis.
• Structural and ionic modulation of Cas12a to enhance SNV discrimination.
• Application of SDS-CRISPR for detecting IDH1 alleles in glioma samples and HIV-1 drug-resistance mutations.
• Integration with lateral-flow strips and AI-assisted smartphone readers for point-of-care testing.

Main Results:

• SDS-CRISPR achieved attomole sensitivity and detected variants at 0.01% frequency for IDH1 alleles.
• On-site detection was achieved within 20 minutes using the integrated system.
• Clinical validation with glioma tissues showed high concordance with immunohistochemistry.
• Plasma cfDNA analysis demonstrated mutation fractions consistent with next-generation sequencing.
• SDS-CRISPR successfully discriminated microRNA isoforms and identified HIV-1 drug-resistance mutations.

Conclusions:

• SDS-CRISPR provides a mechanistically informed and highly precise method for allele discrimination.
• The system offers a universal framework for sensitive molecular diagnostics, applicable to various targets including SNVs, microRNAs, and infectious agents.
• SDS-CRISPR holds significant potential for clinical applications, including intraoperative diagnostics and precision medicine.