CRISPR-Cas12a-Induced DNA Supercoil Relaxation for Nonfluorescent Ratiometric DNA Detection

Introduction:: CRISPR-based diagnostics (CRISPR-Dx) frequently rely on fluorescent, electrochemical, or colorimetric signaling readouts.^1-6 Alternative nonoptical detection method utilizing the size reduction of long λ DNA substrate upon target recognition has the advantages of removing the need for fluorescent labeling and expensive optical measurement instrument.¹ However, precise determination of band shift for long DNA reporters (e.g., λ DNA) requires an intact reference λ DNA reporter run on the same gel, due to the various unexpected interferences on gel band positions such as gel distortion and misalignment. To address this limitation, here we demonstrated a new nonoptical detection mechanism by utilizing CRISPR-Cas12a-induced pUC19 DNA supercoil relaxation to develop a nonfluorescent, ratiometric CRISPR-Cas12a reporting system, which includes an internal reference band for self-calibration. This approach provides a simple yet sensitive DNA sizing-based CRISPR-Dx reporting system that is more suited for rapid point-of-care applications to fight against emerging infectious diseases.

Materials and Methods:: We developed a simple, inexpensive, and nonfluorescent CRISPR-Cas12a-based sensing platform to detect short ssDNA utilizing the conformation change of supercoiled pUC19 DNA into its relaxed circular form. pUC19 is a 2.69-kb circular double-stranded (ds) DNA.⁷ The negatively supercoiled conformation may undergo relaxation and breathing, exposing single-stranded (ss) DNA that could be trans-nicked by LbCas12a.⁸ In this detection platform, we added LbCas12a nuclease, a programmable guide RNA (gRNA), analyte solution (ssDNA target), and pUC19 DNA as nonfluorescent substrate. The CRISPR reaction was performed for 10 mins to ~1 hour at 37˚C, followed by gel electrophoresis analysis to size the reaction products. The extent of conformation change induced by trans-nicking of Cas12a was quantitatively measured by ratiometric analysis of gel electrophoresis images and plotted against target concentrations.


Results, Conclusions, and Discussions:: We found that the nonspecific nicking effect of Cas12a could open the knots of negatively supercoiled pUC19 very fast (less than 10 mins) and turn it into the relaxed conformation (or circular conformation). In the presence of target DNA, we observed that the fraction of supercoiled pUC19 decreased, and the amount of relaxed conformation increased with time (Fig. 1). The relative amount of supercoiled DNA to the relaxed circular DNA was determined by ratiometric intensity analysis of gel electrophoresis bands for target concentration quantification. Further, we tested a few other dsDNA plasmids, such as pBR322, and ΦX174 RF I; and we found that the DNA supercoil relaxation is applicable to many other dsDNAs, which makes the new sensing strategy universal.

As a proof-of-concept, we chose pUC19 as the ratiometric reporter and optimized the reaction conditions (Temperature, pH, buffer type, salt concentrations etc.). We were able to detect ssDNA target with a limit of detection (LOD) of ~2.5 pM without any preamplification step (Figure 2). This simple approach can complement our previous molecular sizing-based CRISPR-Cas12a detection scheme and further enrich the nonoptical signaling mechanism of CRISPR-Dx for potential point-of-use applications.


Acknowledgements (Optional): :

References (Optional): : 1. Mohammad, N.; Katkam, S. S.; Wei, Q., A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double-Stranded λ DNA Reporter. Angewandte Chemie International Edition 2022, 61 (50), e202213920.

2. Chen, J. S.; Ma, E.; Harrington, L. B.; Da Costa, M.; Tian, X.; Palefsky, J. M.; Doudna, J. A., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 2018, 360 (6387), 436-439.

3. Gootenberg, J. S.; Abudayyeh, O. O.; Lee, J. W.; Essletzbichler, P.; Dy, A. J.; Joung, J.; Verdine, V.; Donghia, N.; Daringer, N. M.; Freije, C. A.; Myhrvold, C.; Bhattacharyya, R. P.; Livny, J.; Regev, A.; Koonin, E. V.; Hung, D. T.; Sabeti, P. C.; Collins, J. J.; Zhang, F., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science (New York, N.Y.) 2017, 356 (6336), 438-442.

4. Weng, Z.; You, Z.; Yang, J.; Mohammad, N.; Lin, M.; Wei, Q.; Gao, X.; Zhang, Y., CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics. Angewandte Chemie International Edition 2023.

5. Mohammad, N.; Katkam, S. S.; Wei, Q., Recent Advances in CRISPR-Based Biosensors for Point-of-Care Pathogen Detection. The CRISPR Journal 2022, 5 (4), 500-516.

6. Ackerman, C. M.; Myhrvold, C.; Thakku, S. G.; Freije, C. A.; Metsky, H. C.; Yang, D. K.; Ye, S. H.; Boehm, C. K.; Kosoko-Thoroddsen, T.-S. F.; Kehe, J.; Nguyen, T. G.; Carter, A.; Kulesa, A.; Barnes, J. R.; Dugan, V. G.; Hung, D. T.; Blainey, P. C.; Sabeti, P. C., Massively multiplexed nucleic acid detection with Cas13. Nature 2020, 582 (7811), 277-282.

7. Yanisch-Perron, C.; Vieira, J.; Messing, J., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 1985, 33 (1), 103-119.

8. Murugan, K.; Seetharam, A. S.; Severin, A. J.; Sashital, D. G., CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects. Journal of Biological Chemistry 2020, 295 (17), 5538-5553.