crRNA Truncation Minimizes <i>cis</i> -Cleavage for Universal One-Pot RPA-CRISPR-Cas12a Detection with Attomolar Sensitivity

CRISPR-Cas12a-based diagnostics face a critical challenge in one-pot assays, where cis-cleavage activity prematurely degrades amplification templates, compromising sensitivity. Here, we report a rational design strategy using truncated crRNAs to selectively reduce cis-cleavage while preserving effective trans-cleavage for signal generation. Systematic optimization showed that truncating the crRNA 5′-end by 10–14 nucleotides (+10 Spacer to +6 Spacer) minimizes cis-cleavage while preserving reliable trans-cleavage activity. Mechanistic studies demonstrate that truncated crRNAs reduce binding affinities for both LbCas12a and target dsDNA, effectively disabling template degradation while maintaining signal amplification. Leveraging this principle, we developed a universal one-pot RPA-CRISPR-Cas12a assay with attomolar sensitivity, validated for chikungunya and Epstein–Barr virus detection. Unlike existing approaches requiring additives or external triggers, our method achieves precise activity modulation through crRNA engineering alone, enabling ultrasensitive, one-pot detection of clinical samples with minimal workflow complexity.