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Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection

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Abstract

The efficient signal amplification capacity of several class 2 CRISPR-Cas systems with trans-cleavage activity has exhibited great value in molecular diagnostics, but its potential application for non-nucleic-acid targets is yet underdeveloped. Here, we deploy CRISPR-Cas system for the ultrasensitive detection of protease biomarkers by the coupling of proteolysis-triggered transcription. In this strategy, a protease-activatable RNA polymerase is adopted for the conversion of each protease-catalyzed proteolysis event into the output of multiple programable RNA sequences by in vitro transcription, and the transcribed RNA subsequently serves as the guide RNA of Cas12a proteins with trans-cleavage activity. The rational design of the transcribed RNA efficiently couples the signal conversion and amplification of proteolysis-triggered transcription and the self-signal amplification of CRISPR-Cas12a, resulting in a two-stage amplified detection of target protease. The versatility of this strategy has been demonstrated in the detection of protease biomarkers including MMP-2 and thrombin with femtomolar sensitivity, which is 5–6 orders of magnitude lower than that of the standard peptide-based methods. Moreover, the proposed method has been further applied in the analysis of MMP-2 secreted by different cancer cell lines as well the assessment of MMP-2 activity in clinical serum samples, providing a generic method for the ultrasensitive detection of protease biomarkers in biochemical research and clinical diagnosis.

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References

  1. Siegel RL, Miller KD, Jemal A. CA-A Cancer J Clin, 2016, 66: 7–30

    Google Scholar 

  2. Wu L, Qu X. Chem Soc Rev, 2015, 44: 2963–2997

    CAS  PubMed  Google Scholar 

  3. Sawyers CL. Nature, 2008, 452: 548–552

    CAS  PubMed  Google Scholar 

  4. Zen K, Zhang CY. Med Res Rev, 2012, 32: 326–348

    PubMed  Google Scholar 

  5. López-Otín C, Bond JS. J Biol Chem, 2008, 283: 30433–30437

    PubMed  PubMed Central  Google Scholar 

  6. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DBT, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. Science, 2016, 353: aaf5573

    PubMed  PubMed Central  Google Scholar 

  7. Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA. Science, 2018, 362: 839–842

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang H, Gao P, Rajashankar KR, Patel DJ. Cell, 2016, 167: 1814–1828.e12

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Li SY, Cheng QX, Liu JK, Nie XQ, Zhao GP, Wang J. Cell Res, 2018, 28: 491–493

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Li SY, Cheng QX, Wang JM, Li XY, Zhang ZL, Gao S, Cao RB, Zhao GP, Wang J. Cell Discov, 2019, 5: 17

    PubMed  PubMed Central  Google Scholar 

  11. Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. Science, 2018, 360: 436–439

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Science, 2018, 360: 439–444

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science, 2017, 356: 438–442

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL, MacInnis BL, Bosch I, Gehrke L, Zhang F, Sabeti PC. Science, 2018, 360: 444–448

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Teng F, Guo L, Cui T, Wang XG, Xu K, Gao Q, Zhou Q, Li W. Genome Biol, 2019, 20: 132

    PubMed  PubMed Central  Google Scholar 

  16. Wang B, Wang R, Wang D, Wu J, Li J, Wang J, Liu H, Wang Y. Anal Chem, 2019, 91: 12156–12161

    CAS  PubMed  Google Scholar 

  17. Hu M, Yuan C, Tian T, Wang X, Sun J, Xiong E, Zhou X. J Am Chem Soc, 2020, 142: 7506–7513

    CAS  PubMed  Google Scholar 

  18. Dai Y, Somoza RA, Wang L, Welter JF, Li Y, Caplan AI, Liu CC. Angew Chem Int Ed, 2019, 58: 17399–17405

    CAS  Google Scholar 

  19. Liang M, Li Z, Wang W, Liu J, Liu L, Zhu G, Karthik L, Wang M, Wang KF, Wang Z, Yu J, Shuai Y, Yu J, Zhang L, Yang Z, Li C, Zhang Q, Shi T, Zhou L, Xie F, Dai H, Liu X, Zhang J, Liu G, Zhuo Y, Zhang B, Liu C, Li S, Xia X, Tong Y, Liu Y, Alterovitz G, Tan GY, Zhang LX. Nat Commun, 2019, 10: 3672

    PubMed  PubMed Central  Google Scholar 

  20. Xiong Y, Zhang J, Yang Z, Mou Q, Ma Y, Xiong Y, Lu Y. J Am Chem Soc, 2020, 142: 207–213

    CAS  PubMed  Google Scholar 

  21. Bond JS. J Biol Chem, 2019, 294: 1643–1651

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Lee M, Fridman R, Mobashery S. Chem Soc Rev, 2004, 33: 401–409

    CAS  PubMed  Google Scholar 

  23. Vizovisek M, Vidmar R, Drag M, Fonovic M, Salvesen GS, Turk B. Trends Biochem Sci, 2018, 43: 829–844

    CAS  PubMed  Google Scholar 

  24. Chang C, Werb Z. Trends Cell Biol, 2001, 11: S37–S43

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Deryugina EI, Quigley JP. Cancer Metast Rev, 2006, 25: 9–34

    CAS  Google Scholar 

  26. Wang X, Khalil R A. Adv Pharmacol, 2018, 81: 241–330

    CAS  PubMed  Google Scholar 

  27. Roy R, Yang J, Moses MA. JCO, 2009, 27: 5287–5297

    CAS  Google Scholar 

  28. Qin Y, Yin Y, Liu YK, Feng JJ, Jin LF, Pu Y, Hua D, Qi XW. Cell Biochem Biophys, 2015, 73: 253–259

    CAS  Google Scholar 

  29. Martínez D, Munera M, Cantillo JF, Wortmann J, Zakzuk J, Keller W, Caraballo L, Puerta L. Int J Mol Sci, 2019, 20: 3025

    PubMed Central  Google Scholar 

  30. Mori S, Pawankar R, Ozu C, Nonaka M, Yagi T, Okubo K. Allergy Asthma Immunol Res, 2012, 4: 231–239

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Cheng H, Li SY, Zheng HR, Li CX, Xie BR, Chen KW, Li B, Zhang XZ. Anal Chem, 2017, 89: 4349–4354

    CAS  PubMed  Google Scholar 

  32. Gao X, Guo W, Jiang L, Hu B, Liu X, Xu K, Tang B. Sci China Chem, 2020, 63: 135–140

    CAS  Google Scholar 

  33. Fischbach M, Resch-Genger U, Seitz O. Angew Chem Int Ed, 2014, 53: 11955–11959

    CAS  Google Scholar 

  34. Ma T, Hou Y, Zeng J, Liu C, Zhang P, Jing L, Shangguan D, Gao M. J Am Chem Soc, 2018, 140: 211–218

    CAS  PubMed  Google Scholar 

  35. Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E. Nature, 2016, 532: 517–521

    CAS  PubMed  Google Scholar 

  36. Gao P, Yang H, Rajashankar KR, Huang Z, Patel DJ. Cell Res, 2016, 26: 901–913

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Kundert K, Lucas JE, Watters KE, Fellmann C, Ng AH, Heineike BM, Fitzsimmons CM, Oakes BL, Qu J, Prasad N, Rosenberg OS, Savage DF, El-Samad H, Doudna JA, Kortemme T. Nat Commun, 2019, 10: 2127

    PubMed  PubMed Central  Google Scholar 

  38. Liu F, Yang M, Song W, Luo X, Tang R, Duan Z, Kang W, Xie S, Liu Q, Lei C, Huang Y, Nie Z, Yao S. Chem Sci, 2020, 11: 2993–2998

    CAS  Google Scholar 

  39. Pu J, Chronis I, Ahn D, Dickinson BC. J Am Chem Soc, 2015, 137: 15996–15999

    CAS  PubMed  PubMed Central  Google Scholar 

  40. English MA, Soenksen LR, Gayet RV, de Puig H, Angenent-Mari NM, Mao AS, Nguyen PQ, Collins JJ. Science, 2019, 365: 780–785

    CAS  PubMed  Google Scholar 

  41. Dong D, Ren K, Qiu X, Zheng J, Guo M, Guan X, Liu H, Li N, Zhang B, Yang D, Ma C, Wang S, Wu D, Ma Y, Fan S, Wang J, Gao N, Huang Z. Nature, 2016, 532: 522–526

    CAS  PubMed  Google Scholar 

  42. Swarts DC, van der Oost J, Jinek M. Mol Cell, 2017, 66: 221–233.e4

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV, Ishitani R, Zhang F, Nureki O. Cell, 2016, 165: 949–962

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Sheen-Chen SM, Chen HS, Eng HL, Sheen CC, Chen WJ. Cancer Lett, 2001, 173: 79–82

    CAS  PubMed  Google Scholar 

  45. Murnane MJ, Cai J, Shuja S, McAneny D, Klepeis V, Willett JB. Int J Cancer, 2009, 125: 2893–2902

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Garbisa S, Scagliotti G, Masiero L, Francesco C D, Caenazzo C, Onisto M, Micela M, Stetler-Stevenson W G, Liotta L A. Cancer Res, 1992, 52: 4548–4549

    CAS  PubMed  Google Scholar 

  47. Li L, Lin H, Lei C, Nie Z, Huang Y, Yao S. Biosens Bioelectron, 2014, 54: 42–47

    CAS  PubMed  Google Scholar 

  48. Maragoudakis ME, Tsopanoglou NE, Andriopoulou P, Maragoudakis MEM. Matrix Biol, 2000, 19: 345–351

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21974038, 21725503) and the Fundamental Research Funds for the Central Universities.

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  1. These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, China

    Min Yang, Kai Shi, Fang Liu, Wenyuan Kang, Chunyang Lei & Zhou Nie

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  1. Min Yang
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  2. Kai Shi
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  3. Fang Liu
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  4. Wenyuan Kang
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  5. Chunyang Lei
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Correspondence to Chunyang Lei or Zhou Nie.

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Yang, M., Shi, K., Liu, F. et al. Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection. Sci. China Chem. 64, 330–336 (2021). https://doi.org/10.1007/s11426-020-9863-y

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  • DOI: https://doi.org/10.1007/s11426-020-9863-y

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