DNA extensions near the scaffold improve Cas12a cutting to detect a wide range of nucleic acids directly

Detection limits of 1.3 fM for miRNA and 93 fM for mRNA are achieved using a newly engineered DNA activator architecture.

• The engineered DNA activator architecture enhances Cas12a trans-cleavage activity significantly.
• Appending an overhang at the scaffold-proximal end of the activator increases Cas12a activation, while distal extensions hinder performance.
• This system allows for direct RNA detection without the need for reverse transcription.
• Exceptional sensitivity is demonstrated in detecting various RNA targets, including microRNAs and long mRNAs.
• A Split-PEA format provides high discriminatory power for identifying specific genetic mutations, such as the EGFR T790 M mutation at low frequencies.

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