Building Amplification-Free CRISPR/Cas Biosensors Through Autocatalysis Reaction Induced by Circular DNA Nanostructure

Endonuclease Cas12a, as one key part of the CRISPR/Cas biotechnology, possessed a unique dual enzymatic function which has been widely used for various applications1. However, the limits trans-cleavage efficiency of a single Cas12a ribonucleoprotein (RNP) holds a major barrier for its further growth as a novel biotechnology toolbox2. Unlike any previous studies, we reported the manipulation of Cas12a activity with special circular DNA nanostructure to establish a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system, which is able to break the standard reaction pattern of the Cas12a RNP. In our test, >1,000 Cas12a RNPs can be activated with the presence of a single targeted nucleic acid sequence. We carefully investigated the autocatalysis reaction activity of AutoCAR and also its potential mechanism beneath the changed linear to exponential reaction pattern. In addition, to demonstrate the potential value of AutoCAR in bioanalysis, its autocatalysis reaction can be transferred into a groundbreaking approach to existed CRISPR/Cas12a diagnostics, and realized 1 attomole sensitivity to nucleic acid detections at room temperature without the need for any additional amplifications. Furthermore, 1 attomole RNA detection has also been realized by using modified AutoCAR without the need for reverse transcription. These capabilities have been used to detect ~1 copy/μL DNA/RNA targets from H. pylori or SARS-CoV-2 genomes, respectively. The development of AutoCAR served as an exploration to manipulate the Cas enzyme activity patterns, and revealed the under covered potentials of CRISPR/Cas biotechnology for a boarder range of applications, such as biochemistry, bioanalysis and diagnostic applications.

Learning Objectives: 

1. Define the key factors affect the efficiency of Cas12a autocatalysis reaction.

2. Discuss the advantages and disadvantages of the Cas12a autocatalysis reaction developed here.

3. Evaluate the biosensing performance of AutoCAR for nucleic acid detection.