crRNA Truncation Minimizes cis-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.