Two Ways Extending crRNA Ends Reduces Cas12a Activity for Controlled One-Pot CRISPR Tests

An assay achieved 10M sensitivity and 100% clinical concordance with qPCR using a 7-nucleotide RNA extension.

• Minimal 3'-RNA extensions of 7-13 nucleotides can effectively reduce Cas12a activity.
• The reduction occurs through two mechanisms: competitive cleavage of the RNA extension and blockage of the RuvC catalytic site.
• SMART-POT is a new one-pot detection platform that simplifies the process by avoiding the need for screening noncanonical PAM crRNAs.
• The assay demonstrated perfect specificity against 13 high-risk HPV genotypes.

Simplified