CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells

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.

Keywords: ATP sensing; CRISPR/Cas; biosensor; nanomedicine; nanoneedles; porous silicon; sensor.

Figure 1

Schematic representation of the CRISPR/Cas-assisted nanoneedle sensor for the intracellular detection of ATP. The locked activator-modified nanoneedles are placed in the cell culture well with the nanoneedles facing toward cells (nanoneedles on top interfacing), and the whole setup is centrifuged. The centrifugation interfaces the nanoneedles with the cells, presenting the aptamers in the cytosol where they bind to intracellular ATP. When bound to ATP, the aptamer is released and unlocks the activator. After centrifugation, the nanoneedles are retrieved and incubated with the reporter system containing a Cas12a/crRNA complex and a ssDNA F-Q. The exposed activator triggers the cleavage of ssDNA-FQ by Cas12a/crRNA, yielding a detectable fluorescent signal.

Figure 2

Validation of the nanoCRISPR sensor for ATP detection. (a) Agarose gel electrophoresis analysis of CRISPR/Cas12 activation upon ATP detection by the locked activator in solution. ssDNA without secondary structure was used as the substrate (lane 1: ssDNA, lane 2: ssDNA + Cas12a/crRNA + activator, lane 3: ssDNA + Cas12a/crRNA + locked activator + ATP, lane 4: ssDNA + Cas12a/crRNA + locked activator, and lane 5: ssDNA + Cas12a/crRNA). (b) Representative fluorescence microscopy images of bare nanoneedles (top) and APTES-functionalized nanoneedles following incubation with amine-reactive FITC. Scale bars 6 μm. Images are acquired at 16bit using the same conditions, but fluorescence is reported on different intensity scales, found under the image. (c) Quantification of fluorescence intensity from the microscopy images shown in (b) illustrating APTES conjugation on nanoneedles. Analysis is performed on five images from each of three independent nanoneedle chips. Data are reported as mean with standard deviation and each data point is reported in the graph. Statistical analysis is performed by unpaired t-test. ****p < 0.0001. (d) Representative fluorescence microscopy images of nanoneedles at different stages of functionalization following incubation with FITC-Biotin (FBiotin). Scale bars 6 μm. Images are acquired at 16bit using the same conditions, but fluorescence is reported on different intensity scales, found under the image. (e) Quantification of fluorescence intensity from the microscopy images shown in (d) illustrating successful aptamer conjugation on nanoneedles. The decrease in fluorescence following aptamer conjugation is proportional to the activator occupancy of biotin binding sites. Analysis is performed on five images from each of three independent nanoneedle chips. Data are reported as mean with standard deviation and each data point is reported in the graph. Statistical analysis is performed by 1-way analysis of variance with posthoc Tukey test. ****p < 0.0001. (f,g) SEM assessment of the nanoneedle chip at key stages of the nanoCRISPR assembly process. (f) Quantification of nanoneedles dimensions, and (g) SEM images of nanoneedles. (h) Fluorescent signal generation from the assembled nanoCRISPR sensor upon ATP detection (purple spectrum: activator immobilized on nanoneedles + Cas12a/crRNA + ssDNA F-Q, gray spectrum: bare nanoneedles + Cas12a/crRNA + ssDNA F-Q, orange spectrum: Locked Activator immobilized on nanoneedles + ATP + Cas12a/crRNA + ssDNA F-Q, and blue spectrum: locked activator immobilized on nanoneedles + Cas12a/crRNA + ssDNA F-Q). The gray and blue spectrum overlap in the graph as in both instances no activation is observed.

Figure 3

Performance of the nanoCRISPR sensor for the detection of ATP. (A) Fluorescence response of the nanoCRISPR in the presence of ATP ranging from 0 μM to 200 μM (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μM) (b) linear regression of the fluorescence intensity at 520 nm as a function of ATP concentration. Data represent the average ± standard deviation from five measurements.

Figure 4

Selectivity of nanoCRISPR for ATP detection. (a) Fluorescence spectra recorded from nanoCRISPR after exposure to 200 μM ATP, UTP, CTP, and GTP. (b) Corresponding histograms for the fluorescence intensity at 520 nm as measured from the fluorescence spectra. Data represent mean plus standard deviation from five measurements. Individual measurements are reported in the graph. One way ANOVA with Tukey post-test; ****p < 0.0001.

Figure 5

Intracellular detection of ATP using nanoCRISPR in live cells. (a) Comparison of the fluorescence signal detected at 520 nm for nanoCRISPR interfaced nanoneedles on the bottom. Individual groups include HEK-293 cells at 30k and 50k cell concentration, with and without oligomycin treatment and dead cells, at the two centrifugation speeds of 300 and 600 rpm. (b) Comparison of the fluorescence signal detected at 520 nm for nanoCRISPR interfaced nanoneedles on top. Individual groups include HEK-293 cells at 30k and 50k cell concentration, with and without oligomycin treatment and dead cells, at the two centrifugation speeds of 300 and 600 rpm. Data represent mean plus standard deviation from five measurements. Statistical analysis was performed by two-way analysis of variance with the Holm-Sidak multiple-comparison test. *p < 0.0332, **p < 0.021, ***p < 0.0002, and ****p < 0.0001. (c) LIVE/DEAD assay on 50k HEK-293 cells following nN-T interfacing with nanoCRISPR at 300 rpm. Left to right: bright field; green fluorescence from Calcein AM indicating live cells; red fluorescence from EthD-III indicating dead cells; merged channels. Scale bar is 100 μm.