Conformational transformation of activator strand directly regulates CRISPR/Cas12a system activity for live cell sensing of multiple biomolecules

•The function of the activator strand can be adjusted without the need for additional auxiliary probes, thereby directly regulating CRISPR/Cas12a system activity, simplifying experimental operations and reducing design costs.•Due to the advantage of a low background, this CRISPR/Cas12a-based sensor has been proven to be well used for precise imaging analysis of targets in living cells.•By designing different target recognition sequences, the sensing and detection of different target molecules (ATP and nucleolin) were realized.•The biosensor can be combined with lateral flow assay (LFA) to enable rapid detection of targets and generate visualization results in minutes showing potential point-of-care (POC) or field test application capabilities. In recent years, biosensors designed based on the CRISPR/Cas12a system have opened up a new era in biosensing. However, the activator strand in the existing CRISPR/Cas12a-based sensing strategies are usually designed with 20 bases complementary to crRNA, it is difficult to completely shut down the trans-cleavage activity of the CRISPR/Cas12a system. To address this issue and simultaneously construct a biosensor with an “on-off-on” mode, we shortened the activator strand complementary to crRNA to 16 bases and embedded it into a hairpin structure depriving its ability to activate the Cas12a. We all know that the binding of aptamers with small molecules involves folding into a complex structure that is complementary to the surface of small molecules, thus forming a stable complex. To further expand the application of the CRISPR/Cas12a system in the field of biosensors, we selected small-molecule ATP and nucleolin as targets to confirm that the formation of the complex three-dimensional structure at the 3′- end of activator strand does not affect the activity of CRISPR/Cas12a system. Therefore, by designing only one allosteric activator strand, it is possible to regulate CRISPR/Cas12a system’s activity. This design not only reduces the number of DNA strands used in the sensing system, but also eliminates the need for complex concentration optimization operations. Furthermore, the stability of the intramolecular hairpin structure can minimize fluctuations in the background signal, ensuring stable and low-background signals in live cell applications, this further expands the toolbox of applications for the CRISPR/Cas12a system.