Precise detection of single-letter DNA mutations using a CRISPR tool with two targeting guides and staggered cutting

A detection limit as low as 10¹ CFU/mL¹ CFU/mL** is achieved using a new sensing strategy for single-nucleotide mutations.

• The STAND strategy employs a dual-guide RNA system to enhance the specificity of single-nucleotide mutation discrimination.
• Cis-staggered cleavage of double-stranded DNA generates a sticky-end intermediate that is recognized by a secondary guide RNA.
• This method allows the conversion of single-nucleotide variations into amplified fluorescence signals with high fidelity.
• STAND demonstrates superior accuracy in distinguishing clinically relevant targets compared to conventional qPCR.
• The approach's modular design and minimal guide requirements suggest potential for broader applications in genetic analysis.

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