CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells, 2023, Hongki Kim et al

Abstract


The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging.

Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability.

We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

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Conclusions
We developed nanoCRISPR as an intracellular ATP sensing platform, combining nanoneedle technology for intracellular access with aptamer ATP recognition and CRISPR/Cas12a amplification. We assembled on nanoneedles a locked-activator sensor capable of selectively recognizing ATP, resulting in unlocking of the Cas12a activator and triggering the cleavage of ssDNA-FQ by Cas12a, yielding a detectable fluorescent signal. NanoCRISPR can quantitatively detect ATP with an LOD of 246 nM and high selectivity against other triphosphate nucleotides.

Moreover, by nanoneedle on top interfacing with the assistance of centrifugation, nanoCRISPR can detect intracellular ATP in living cells in 30 min. The sensor can discriminate between different intracellular ATP concentrations and different cell numbers without impacting cell viability.

Future studies should systematically evaluate the variations in ATP detection performance due to nonspecific interactions and cross-reactivity within the complex intracellular environment. This approach opens the way to noninvasive longitudinal monitoring of cell viability for long-term culture and monitoring of the effects of challenges to cells. It is anticipated that the simple, rapid, and sensitive nanoCRISPR sensor can be applied in biomedical studies and cancer research for monitoring dynamic ATP levels and extended for detecting a range of other analytes in living sample.