Direct Detection of MiRNAs miR-34a, -145, and -218 with CRISPR/Cas13a-nuclease

Article Sidebar

pdf html (Русский) html
Published: Dec 7, 2023
DOI: https://doi.org/10.18097/BMCRM00203
Keywords:
microRNA; detection; CRISPR/Cas-nuclease

Main Article Content

O.S. Timoshenko
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia
L.K. Kurbatov
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia
S.A. Khmeleva
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia
K.G. Ptitsyn
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia
S.P. Radko
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia
A.V. Lisitsa
Institute of Biomedical Chemistry, 10 Pogodinskaya st., Moscow, 119121 Russia

Abstract

Using CRISPR/Cas13a-nuclease we have demonstrated a feasibility of direct detection of three miRNAs, miR-34a, -145, and -218 (their molecular signature is suggested as a diagnostic and prognostic biomarker in cervical cancer),. The detection is based on registration of a cleavage of molecular reporters bearing a fluorophore and a quencher by the complex of CRISPR/Cas13a-nuclease and guide RNA (gRNA) with a spacer of 21-23 nucleotides long. The detection sensitivity varied among miRNAs tested by 10-fold, presumably due to the unwanted intramolecular partial base paring of gRNA. The miRNA detection with Cas13a nuclease strongly depended on the presence of background RNA thus potentially compromising its direct application to complex media in a general case. Further optimization of measurement conditions including probably an additional amplification of the signal generated by collateral activity of Cas13a nuclease is necessary to directly detect miR-34a, -145, and -218 in biological samples.

Article Details

How to Cite
Timoshenko, O., Kurbatov, L., Khmeleva, S., Ptitsyn, K., Radko, S., & Lisitsa, A. (2023). Direct Detection of MiRNAs miR-34a, -145, and -218 with CRISPR/Cas13a-nuclease. Biomedical Chemistry: Research and Methods, 6(4), e00203. https://doi.org/10.18097/BMCRM00203
Issue
Vol. 6 No. 4 (2023)
Section
EXPERIMENTAL RESEARCH

References

  1. Cai, Y., Yu, X., Hu, S., Yu, J. (2009) A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics, 7(4), 147-154. DOI
  2. Saliminejad, K., Khorram Khorshid, H.R., Soleymani Fard, S., Ghaffari, S.H. (2019) An overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol., 234(5), 5451-5465. DOI
  3. Macfarlane, L.A., Murphy, P.R. (2010) MicroRNA: Biogenesis, Function and Role in Cancer. Curr Genomics., 11(7), 537-561. DOI
  4. de Planell-Saguer, M., Rodicio, M.C. (2013) Detection methods for microRNAs in clinic practice Clin Biochem. 46(10-11), 869-878. DOI
  5. Aftab, M., Poojary, S.S., Seshan, V., Kumar, S., Agarwal, P., Tandon, S., Zutshi, V., Das, B.C. (2021) Urine miRNA signature as a potential non-invasive diagnostic and prognostic biomarker in cervical cancer. Sci Rep., 11(1),10323. DOI
  6. Kiseleva, Y.Y., Ptitsyn, K.G., Radko, S.P., Zgoda, V.G., Archakov, A.I. (2016) Digital droplet PCR - a prospective technological approach to quantitative profiling of microRNA. Biomeditsinskaya Khimiya. 62(4), 403-410. DOI
  7. Ferreira, C.E.S., Guerra, J.C.C., Slhessarenko, N., Scartezini, M., Franca, C.N., Colombini, M.P., Berlitz, F., Machado, A.M.O., Campana, G.A., Faulhaber, A.C.L., Galoro, C.A., Dias, C.M., Shcolnik, W., Martino, M.D.V., Cesar, K.R., Sumita, N.M., Mendes, M.E., Faulhaber, M.H.W., Pinho, J.R.R., Barbosa, I.V., Batista, M.C., Khawali, C., Pariz V.M., Andriolo, A. (2018) Point-of-Care Testing: General Aspects. Clin Lab., 64(1), 1-9. DOI
  8. Kaminski, M.M., Abudayyeh, O.O., Gootenberg, J.S., Zhang, F., Collins, J.J. (2021) CRISPR-based diagnostics. Nat Biomed Eng., 5(7), 643-656. DOI
  9. Wu, W.Y., Lebbink, J.H.G., Kanaar, R., Geijsen, N., van der Oost (2018) Genome editing by natural and engineered CRISPR-associated nucleases. J. Nat Chem Biol. 14(7), 642-651. DOI
  10. Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D.B., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander, E.S., Koonin, E.V., Zhang, F. (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 353(6299), aaf5573. DOI
  11. Shan, Y., Zhou, X., Huang, R., Xing, D. (2019) High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity. Anal Chem., 91(8), 5278-5285. DOI
  12. 12. Wang, J.Y., Chen, L.J. (2019) The role of miRNAs in the invasion and metastasis of cervical cancer. Biosci Rep., 39(3), BSR20181377. DOI
  13. Kurbatov, L.K., Radko, P. S., Kravchenko, S.V., Kiseleva, O.I., Durmanov, N.D., Lisitsa ,A.V. (2020) Single Stage Purification of CRISPR/Cas13a Nuclease by Metal-Chelating Chromatography Following Heterologous Expression with Preservation of Collateral Ribonuclease Activity. Applied Biochemistry and Microbiology, 56(6), 671-677 DOI
  14. Kurbatov, L., Radko, S., Khmeleva, S., Timoshenko, O., & Lisitsa, A. (2022). Standardization of Recombinant CRISPR/Cas13a-nuclease Preparations by Using RNase A of Known Activity.. Biomedical Chemistry: Research and Methods, 5(4), e00177. DOI
  15. Gu, S., Zhang, Y., Jin, L., Huang, Y., Zhang, F., Bassik, M.C., Kampmann, M., Kay, M.A. (2014) Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic Acids Res., 42(19), 12169-12176. DOI
  16. Seok, H., Lee, H., Jang, E.S., Chi, S.W.(2018) Evaluation and control of miRNA-like off-target repression for RNA interference. Cell Mol Life Sci., 75(5), 797-814. DOI
  17. Bruch, R., Baaske, J., Chatelle, C., Meirich, M., Madlener, S., Weber W., Dincer, C., Urban, G.A. (2019) CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics. Adv. Mater., 31(51), e1905311. DOI
  18. Bruch, R., Johnston, M., Kling, A., Mattmüller, T., Baaske, J., Partel, S., Madlener, S., Weber, W., Urban, G.A., Dincer, C.(2021) CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics. Biosens Bioelectron., 177, 112887. DOI
  19. Cui, Y., Fan, S., Yuan, Z., Song, M., Hu, J., Qian, D., Zhen, D., Li, J., Zhu, B. (2021) Ultrasensitive electrochemical assay for microRNA-21 based on CRISPR/Cas13a-assisted catalytic hairpin assembly Talanta. , 224:121878. DOI
  20. Zhou, T., Huang, R., Huang, M., Shen, J., Shan, Y., Xing, D. (2020) CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection. Adv Sci (Weinh), 7(13), 1903661. DOI
  21. Sha, Y., Huang, R., Huang, M., Yue, H., Shan, Y., Hu. J., Xing, D. (2021) Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity. Chem Commun (Camb)., 57(2), 247-250. DOI
  22. Zhao, D., Tang, J., Tan, Q., Xie, X., Zhao, X., Xing, D. (2023) CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection. Talanta., 260, 124582. DOI