Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

Abstract Trans -cleavage rates of Cas endonucleases such as Cas12a remain limited despite optimisation efforts. We found that Cas12a ribonucleoproteins poorly bind to, and are only minimally activated by small circular DNA nanostructures which partially match the gRNA sequence. However, activation is restored when the nanostructure is linearised. Building on this finding, we established a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system which allows a single nucleic acid target to activate multiple ribonucleoproteins, and increase the achievable trans -cleavage rates per target by over 3 orders of magnitude. A rate equation-based model was built to interpret the observed nonlinear, near-exponential rate trends. The AutoCAR system was applied for genomic DNA ( H. Pylori ) and RNA (SARS-CoV-2) diagnostics at 1 aM level sensitivity (1.4 copies/µL and 0.36 copies/µL, respectively), comparable to the polymerase chain reaction without any additional signal amplification at room temperature. The RNA detection with Cas12a RNPs did not require reverse transcription.