OPTIMIZATION OF CRISPR/CAS12A TRANS-CLEAVAGE REACTION FOR DETECTION OF THE STAPHYLOCOCCUS AUREUS NUCA GENE
Institute of Tropical Medicine, Joint Vietnam-Russia Tropical Science and Technology Research Center
63 Nguyen Van Huyen, Nghia Do, Cau Giay, Ha Noi
Phone: +84974282734; Email: doai.vn@gmail.com
Main Article Content
Abstract
Staphylococcus aureus (S. aureus) is the most prevalent pathogen within the staphylococcal group, classified as a gram-positive bacterium responsible for severe infections affecting the skin, bloodstream, organs, and gastrointestinal tract. Consequently, the development of early and accurate detection tools is a research priority. Recently, CRISPR/Cas technology, particularly Cas12a, has emerged as a promising diagnostic tool due to its ability to trans-cleave single-stranded DNA (ssDNA) following the specific cleavage of double-stranded DNA (dsDNA) targets. In this study, we optimized a CRISPR/Cas12a trans-cleavage reaction for detecting the nucA gene, a specific marker of S. aureus. To enhance enzyme activity, four crRNAs (crRNA1–4) were designed and tested, each exhibiting different effects on the reaction. However, a mixture of three crRNAs demonstrated higher efficiency than reactions using a single crRNA. Additionally, key reaction parameters, including enzyme concentration, additives, probe sequence, and incubation temperature, were systematically evaluated. The optimized reaction conditions included 6.25 nM LbCas12a combined with the mixture of three crRNAs (crRNA1–3) in a reaction containing 10 mM dithiothreitol (DTT) and 1 µM TA5C ssDNA FAM-BHQ1 probe. The assay was incubated at 39°C for 30 minutes, achieving a detection limit of 284.8 amol or 1.72 × 10⁸ copies of the nucA gene. Fluorescent signals were detectable under a standard UV transilluminator. This study provides valuable insights for the development of CRISPR-based diagnostic kits for S. aureus detection.
http://doi.org/10.58334/vrtc.jtst.n38.01
Keywords
Staphylococcus aureus, nucA, CRISPR/Cas12a, trans-cleavage, diagnostic
Article Details
References
2. G.Y.C. Cheung, J.S. Bae and M. Otto, Pathogenicity and virulence of Staphylococcus aureus, Virulence, Vol.12, pp. 547–569, 2021. DOI: 10.1080/21505594.2021.1878688.
3. A.G. Jensen et al. , risk factors for hospital-acquired Staphylococcus aureus bacteremia, Arch. Intern. Med., Vol. 159, pp. 1437, 1999. DOI:10.1001/archinte.159.13.1437
4. A. Sanchini, Recent developments in phenotypic and molecular diagnostic methods for antimicrobial resistance detection in Staphylococcus aureus: A Narrative Review, Diagnostics, Vol.12, pp. 208, 2022. DOI: 10.3390/diagnostics12010208
5. F.-J. Schmitz, et al., Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR, Journal of Medical Microbiology, Vol.46, pp. 773–778, 1997. DOI: 10.1099/00222615-46-9-773
6. W.J. Mason, J.S. Blevins, K. Beenken, N. Wibowo, N. Ojha and M.S. Smeltzer, Multiplex PCR protocol for the diagnosis of Staphylococcal infection, J. Clin. Microbiol., Vol.39, pp. 3332–3338, 2001. DOI: 10.1128/JCM.39.9.3332-3338
7. J. Tang et al., The staphylococcal nuclease prevents biofilm formation in Staphylococcus aureus and other biofilm-forming bacteria, Sci. China Life Sci., Vol.54, pp. 863–869, 2011. DOI: 10.1007/s11427-011-4195-5
8. K. Tam, V.J. Torres, Staphylococcus aureus secreted toxins and extracellular enzymes, Microbiol. Spectr., Vol.7, pp. 7.2.16, 2019. DOI: 10.1128/microbiolspec.GPP3-0039-2018
9. R. Barrangou, The roles of CRISPR–Cas systems in adaptive immunity and beyond, Current Opinion in Immunology, 2015. Vol.32, pp. 36–41. DOI: 10.1016/j.coi.2014.12.008
10. G.A. Heinz, M.-F. Mashreghi, CRISPR-Cas-System als molekulare schere für gentherapie, Z. Rheumatol., Vol.76, pp. 46–49, 2017. DOI: 10.1007/s00393-017-0267-7
11. E. Tóth et al., Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases, Nucleic Acids Research, Vol.48 ,pp. 3722–3733, 2020. DOI: 10.1093/nar/gkaa110
12. S.-Y. Li, Q.-X. Cheng, J.-K. Liu, X.-Q. Nie, G.-P. Zhao and J. Wang, CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA, Cell Res, Vol.28, pp. 491–493, 2018. DOI: 10.1038/s41422-018-0022-x
13. J.S. Chen et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science, Vol.360, pp.436–439, 2018. DOI: 10.1126/science.aar6245
14. X. Cao et al., Cas12a/guide RNA-based platforms for rapidly and accurately identifying Staphylococcus aureus and methicillin-resistant S. aureus, Microbiol. Spectr. , Vol.11, pp. e04870-22, 2023. DOI: 10.1128/spectrum.04870-22
15. D. Xu, H. Zeng, W. Wu, H. Liu and J. Wang, Isothermal amplification and CRISPR/Cas12a-system-based assay for rapid, sensitive and visual detection of Staphylococcus aureus, Foods, Vol.12, pp. 4432, 2023. DOI: 10.3390/foods12244432
16. M. Zeinulin, M. Amanzholova, A. Shaizadinova and S. Abeldenov, Advancement in Staphylococcus aureus detection using a RPA-CRISPR-Cas12a fluorescent assay technology, Eurasian Journal of Applied Biotechnology,pp. 23–28, 2023. DOI: 10.11134/btp.3.2023.3
17. Y. Liu et al., One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus, Analytica. Chimica. Acta., Vol.1278 , pp. 341757, 2023.DOI: 10.1016/j.aca.2023.341757
18. S. Bae, J. Park and J.-S. Kim, Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases, Bioinformatics, Vol.30, pp. 1473–1475, 2014. DOI: 10.1093/bioinformatics/btu048
19. H. Lv et al., Definition of CRISPR Cas12a trans-cleavage units to facilitate CRISPR diagnostics, Front. Microbiol, Vol.12, pp. 766464, 2021. DOI: 10.3389/fmicb.2021.766464
20. S. Lee et al., Highly efficient DNA reporter for CRISPR/Cas12a-based specific and sensitive biosensor, BioChip J., Vol.16, pp. 463–470, 2022. DOI: 10.1007/s13206-022-00081-0
21. Y. Li et al., Rapid one-tube RPA-CRISPR/Cas12 detection platform for methicillin-resistant Staphylococcus aureus, Diagnostics, Vol.12 , pp. 829, 2022. DOI: 10.3390/diagnostics12040829
22. Y. Chen et al., Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration, Analytica Chimica Acta, Vol.1231, pp. 340417, 2022. DOI: 10.1016/j.aca.2022.340417
23. F. Deng, Y. Li, B. Li and E.M. Goldys, Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: Application to amplified nucleic acid detection, Sensors and Actuators B: Chemical, Vol.373, pp. 132767, 2022. DOI: 10.1016/j.snb.2022.132767.
24. A.A. Malzahn et al., Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis, BMC Biol., Vol.17, pp. 9, 2019. DOI: 10.1186/s12915-019-0629-5
25. R.T. Fuchs et al., Characterization of Cme and Yme thermostable Cas12a orthologs, Commun. Biol., Vol.5, pp. 325, 2022. DOI: 10.1038/s42003-022-03275-2
26. H. Li et al., Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives, Chem. Soc. Rev., Vol.52, pp. 361–382, 2023. DOI: 10.1039/D2CS00594H
27. L.T. Nguyen, B.M. Smith and P.K. Jain, Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection, Nat. Commun., Vol.11, pp. 4906, 2020. DOI: 10.1038/s41467-020-18615-1
28. K. Shi et al., A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics, Sci. Adv., Vol.7, pp.eabc7802, 2011. DOI: 10.1126/sciadv.abc7802
29. H. Sun et al., A programmable sensitive platform for pathogen detection based on CRISPR/Cas12a -hybridization chain reaction-poly T-Cu, Analytica Chimica Acta, Vol.1317, pp.342888, 2024. DOI: 10.1016/j.aca.2024.342888