SDS‐CRISPR for Single‐Nucleotide Variant Detection

ABSTRACT The CRISPR‐Cas12a system offers a promising platform for simple and sensitive nucleic acid diagnostics, including tumor‐associated variant detection and infectious agent identification. However, its intrinsic mismatch tolerance limits its ability to accurately detect single‐nucleotide variants (SNVs). Here, we introduce S tructure‐ D isruption‐ S ensitive CRISPR (SDS‐CRISPR), a programmable CRISPR‐Cas12a approach that achieves highly precise allele discrimination. Guided by AlphaFold3 modeling and bioinformatic analysis, we uncover how split structural design and ionic modulation reconfigure Cas12a conformations, elucidating the structural basis of SNV discrimination in SDS‐CRISPR. We apply SDS‐CRISPR to detect IDH1 WT and IDH1 R132H alleles with attomole sensitivity and 0.01% variant frequency. To facilitate intraoperative use, we combine SDS‐CRISPR with a lateral‐flow strip and an artificial intelligence‐assisted smartphone reader, enabling on‐site detection within 20 min. Clinical validation with 43 glioma tissue samples shows high concordance with immunohistochemistry, while plasma cfDNA testing demonstrates mutation fractions consistent with next‐generation sequencing. Beyond glioma, SDS‐CRISPR generalizes across molecular targets, discriminating microRNA isoforms and identifying HIV‐1 drug‐resistance mutations. Together, these results establish SDS‐CRISPR as a universal, mechanistically informed, and clinically actionable framework for precision molecular diagnostics.