Development of a multifunctional nucleic acid response platform utilizing the Cas12a and Cas13a integrated targeting system

Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system has ushered in a transformative era in biodetection, leveraging its simplicity and efficiency to enable Cas protein-based signaling systems for applications in early tumor screening, viral detection, and molecular logic circuits. However, the constrained compatibility of CRISPR/Cas-based signaling systems with diverse input types limits their versatility, primarily due to the restricted activation mechanisms of the Cas protein. Herein, we developed the Cas12a and Cas13a Integrated Targeting (CACIT) system, which harnesses DNA/RNA strand displacement reactions to integrate the enzymatic capabilities of Cas12a and Cas13a. This system supports simultaneous DNA and RNA inputs, offering exceptional programmability and cost-effectiveness. By employing strand displacement reactions, the CACIT system achieves synchronized activation of Cas12a and Cas13a. We have demonstrated that the CACIT system excels in single-nucleotide-variant (SNV) detection, viral RNA detection, machine learning-driven nucleic acid concentration response modeling, logic operations, and intracellular imaging. As a streamlined and versatile signaling platform, the CACIT system expands the scope of CRISPR/Cas activation strategies. With its inherent simplicity and compatibility, this system facilitates integration with diverse nanodevices. Further, this system provides a highly programmable, multifunctional computational module for molecular networks, heralding new possibilities for artificial signaling systems.