Precise placement of split CRISPR activators improves sensitive detection of microRNA and classification of cancer cells

Attomolar microRNA detection was achieved with a limit of detection of 112 aM, approximately 480-fold higher sensitivity than intact activators.

• Over 200 split DNA activator configurations were systematically mapped to understand their effects on trans-cleavage activity.
• Nicks introduced in the target strand suppressed activity, with the severity depending on the position of the nick.
• Nicks in the non-target strand enhanced activity.
• An optimized split activator pair was engineered based on these findings, resulting in significantly improved sensitivity.
• The enhanced sensitivity allows for multiplexed profiling of five miRNAs, aiding in machine learning-based cancer cell classification.
• The methodology is applicable to non-nucleic acid targets, demonstrating versatility in diagnostic and biosensing applications.

Simplified