Abstract
Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.
Highlights
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The free doublestranded DNA (dsDNA) bound to the Cas12a–CRISPR RNA (crRNA) complex, activating the nonspecific single-stranded DNA (ssDNA) trans cleavage activity of Cas12a
When we examined the relationship between the slope of the fluorescence intensity and the dsDNA concentration (Supplementary Fig. 10B, 11B), the cleavage rate shows an excellent linear pattern (R2 > 0.99) as the dsDNA concentration increased in the range 5–150 pM (dsDNA(HucR)) or 1–25 pM (dsDNA(TetR)) (Fig. 4a(R), Supplementary Fig. 11C)
Summary
General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. We developed a bacterial-aTF-based in vitro biosensing platform, aTF-NAST (aTF-based nicked DNA-template-assisted signal transduction) by exploiting the competition between T4 DNA ligase and aTFs in binding to nicked DNA4,5 This is a relatively time-consuming, inconvenient and costly method of detecting small molecules. Unlike CRISPR/Cas[9] proteins, the Cas12a endonucleases only require CRISPR RNA (crRNA), but not trans-activating crRNA (tracrRNA), as their guide They recognize a T-rich protospacer-adjacent motif (PAM) instead of a G-rich PAM and generate dsDNA breaks with staggered 5′ ends[6]. The trans cleavage activity of CRISPR-Cas12a on single-stranded DNA (ssDNA) has recently been reported[12,13] This unique property of Cas12a has since been applied to the detection of nucleic acids and offers a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostic applications[13,14]. Cas12a itself seems incapable of detecting small molecules (or molecules with low molecular weights) in fields such as disease diagnosis, food quality control, environmental pollutant detection and metabolic engineering[15,16]
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