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Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors

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Abstract

An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N–C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 − 2000.0 pM and a LOD of 0.5 pM. The discovery of “enhancement and inactivation effect” not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.

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References

  1. Li Q, Song ZL, Zhang YX, Zhu LN, Yang Q, Liu XF, Sun XF, Chen XX, Kong RM, Fan GC, Luo XL (2023) Synergistic Incorporation of two ssDNA activators enhances the trans-cleavage of CRISPR/Cas12a. Anal Chem 95:8879–8888

    Article  CAS  PubMed  Google Scholar 

  2. Ma JY, Wang SY, Du YC, Wang DX, Tang AN, Wang J, Kong DM (2022) “RESET” effect: random extending sequences enhance the trans cleavage activity of CRISPR/Cas12a. Anal Chem 94:8050–8057

    Article  CAS  PubMed  Google Scholar 

  3. Li BZ, Shao ZC, Chen Y (2021) An exonuclease protection and CRISPR/Cas12a integrated biosensor for the turn-on detection of transcription factors in cancer cells. Anal Chim Acta 1165:338478

    Article  CAS  PubMed  Google Scholar 

  4. Lee I, Kwon SJ, Sorci M, Heeger PS, Dordick JS (2021) Highly sensitive immuno-CRISPR assay for CXCL9 detection. Anal Chem 93:16528–16534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Christopher WS, Mahera JK, Nidhi N, Mehmet VY (2021) Recognition of DNA target formulations by CRISPR-Cas12a using a dsDNA reporter. ACS Synth Biol 10:1785–1791

    Article  Google Scholar 

  6. Fu XY, Shi YY, Peng FQ, Zhou M, Yin Y, Tan Y, Chen M, Yin X, Ke GL, Zhang XB (2021) Exploring the trans-cleavage activity of CRISPR/Cas12a on gold nanoparticles for stable and sensitive biosensing. Anal Chem 93:4967–4974

    Article  CAS  PubMed  Google Scholar 

  7. Li JC, Luo T, He Y, Liu H, Deng ZW, Bu JQ, Long X, Zhong S, Yang YJ (2022) Discovery of the Rnase activity of CRISPR–Cas12a and its distinguishing cleavage efficiency on various substrates. Chem Commun 58:2540–2543

    Article  CAS  Google Scholar 

  8. Yuan CQ, Tian T, Sun J, Hu ML (2020) Universal and naked-eye gene detection platform based on the clustered regularly interspaced short palindromic repeats/Cas12a/13a system. Anal Chem 92:4029–4037

    Article  CAS  PubMed  Google Scholar 

  9. Liu ZB, Chen SS, Liu BW, Wu JP, Zhou YB, He LY (2014) Intracellular detection of ATP using an aptamer beacon covalently linked to graphene oxide resisting nonspecific probe displacement. Anal Chem 86:12229–12235

    Article  CAS  PubMed  Google Scholar 

  10. Lee J, Yim Y, Kim S, Choi MH, Choi BS, Lee Y, Min DH (2016) In-depth investigation of the interaction between DNA and nano-sized graphene oxide. Carbon 97(92):98

    Google Scholar 

  11. Zhou ZH, Liu Z, Zhang H, Li CX, Deng KQ (2022) Ψ-type hybridization and CRISPR/Cas12a-based two-stage signal amplification for microRNA detection. Sensor Actuat B-Chem 371:132535

    Article  CAS  Google Scholar 

  12. Xiao J, Liu Z, Li CX, Wang JL, Huang HW, Yi QF, Deng KQ, Li XF (2021) Tunable graphdiyne for DNA surface adsorption: affinities, displacement, and applications for fluorescence sensing. Anal Bioanal Chem 413:3847–3859

    Article  CAS  PubMed  Google Scholar 

  13. Chhowalla M, Shin HS, Eda G, Li LJ, Loh KP, Zhang H (2013) The chemistry of two-dimensional layered transition metal dichalcogenide nanosheets. Nat Chem 5:263–275

    Article  PubMed  Google Scholar 

  14. Li BL, Zou HL, Lu L, Yang Y, Lei JL, Luo HQ (2015) Size-dependent optical absorption of layered MoS2 and DNA oligonucleotides induced dispersion behavior for label-free detection of single-nucleotide polymorphism. Adv Funct Mater 25:3541–3550

    Article  CAS  Google Scholar 

  15. Chandrasekar N, Steffi AP, Ramachandran B, Hwang MT, Faramarzi V, Govarthanan M (2023) MXenes–versatile 2D materials for identification of biomarkers and contaminants in large scale environments–A review. Environ Res 228:115900

    Article  CAS  PubMed  Google Scholar 

  16. Liu BW, Salgado S, Maheshwari V, Liu JW (2016) DNA adsorbed on graphene and graphene oxide: fundamental interactions, desorption and applications. Curr Opin Colloid Interface Sci 26:41–49

    Article  CAS  Google Scholar 

  17. Lu C, Liu YB, Ying YB, Liu JW (2017) Comparison of MoS2, WS2, and graphene oxide for DNA adsorption and sensing. Langmuir 33:630–637

    Article  PubMed  Google Scholar 

  18. Zhang F, Wang SY, Liu JW (2019) Gold nanoparticles adsorb DNA and aptamer probes too strongly and a comparison with graphene oxide for biosensing. Anal Chem 91:14743–14750

    Article  CAS  PubMed  Google Scholar 

  19. Lu C, Huang PJ, Liu BW, Ying YB, Liu JW (2016) Comparison of graphene oxide and reduced graphene oxide for DNA adsorption and sensing. Langmuir 32:10776–10783

    Article  CAS  PubMed  Google Scholar 

  20. Wang H, Ma K, Xu B, Tian WJ (2016) Tunable supramolecular interactions of aggregation-induced emission probe and graphene oxide with biomolecules: an approach toward ultrasensitive label-free and “turn-on” DNA sensing. Small 12:6613–6622

    Article  CAS  PubMed  Google Scholar 

  21. Zhou ZH, Zhang H, Qian XM, Li CX, Deng KQ (2023) The composite of bismuth oxyiodide-bismuth/nitrogen-doped carbon for photoreduction and electrochemical/photoelectrochemical dual-model sensing of Cr(VI). Anal Chim Acta 1253:341092

    Article  CAS  PubMed  Google Scholar 

  22. Ma ZJ, Zhang XL, Wu DP, Han XY, Zhang LM, Wang HJ, Xu F, Gao ZY, Jiang K (2020) Ni and nitrogen-codoped ultrathin carbon nanosheets with strong bonding sites for efficient CO2 electrochemical reduction. J Colloid Interface Sci 570:31–40

    Article  CAS  PubMed  Google Scholar 

  23. Atchudan R, Edison T, Aseer K, Perumal S, Karthik N, Lee Y (2018) Highly fluorescent nitrogen-doped carbon dots derived from Phyllanthus acidus utilized as a fluorescent probe for label-free selective detection of Fe3+ ions, live cell imaging and fluorescent ink. Biosens Bioelectron 99:303–311

    Article  CAS  PubMed  Google Scholar 

  24. Lu LM, Zhang YH, Chen ZS, Feng F, Ma ZQ, Zhang ST, Teng KX, An Q (2021) Elemental diversity-enhanced HER and OER photoelectrochemical catalytic performance in FeCo-AuNP/nitrogen-carbon composite catalysts. Appl Surf Sci 568:151005

    Article  CAS  Google Scholar 

  25. Ding LJ, Zhang ZZ, Wen ZR, You FH, Hao N, Wei J, Qian J, Wang K (2022) 2D/2D heterojunction of ZnIn2S4/N-doped graphene nanosheets for off-type high-performance photoelectrochemical aptasensor. Sensor Actuat B-Chem 367:132033

    Article  CAS  Google Scholar 

  26. Liu XY, Wang H, Deng KQ, Kwee S, Huang HW, Tang L (2019) Single primer based multisite strand displacement reaction amplification strategy for rapid detection of terminal deoxynucleotidyl transferase activity. Anal Chem 91(7482):7486

    Google Scholar 

  27. Ohmichi T, Nakano SI, Miyoshi D, Sugimoto N (2002) Long RNA dangling end has large energetic contribution to duplex stability. J Am Chem Soc 124:10367–10372

    Article  CAS  PubMed  Google Scholar 

  28. Li BZ, Xia AQ, Zhang SL, Suo TY, Ma YJ, Huang H, Zhang X, Chen Y, Zhou XM (2021) A CRISPR-derived biosensor for the sensitive detection of transcription factors based on the target-induced inhibition of Cas12a activation. Biosens Bioelectron 173:112619

    Article  CAS  Google Scholar 

  29. Lu SH, Tong XH, Han Y (2022) Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a. Nat Biomed Eng 6:286–297

    Article  CAS  PubMed  Google Scholar 

  30. Tang C, Wang B, Wang HF, Zhang Q (2017) Defect engineering toward atomic Co-Nx-C in hierarchical graphene for rechargeable flexible solid Zn-air batteries. Adv Mater 29:1703185

    Article  Google Scholar 

  31. Wan KF, Long GY, Liu M, Liang ZX, Tsiakaras P (2015) Nitrogen-doped ordered mesoporous carbon: synthesis and active sites for electrocatalysis of oxygen reduction reaction. Appl Catal B 165:566–571

    Article  CAS  Google Scholar 

  32. Liu LL, Wu DH, Zhang L, Feng JJ, Wang AJ (2023) FeCo alloy entrapped in N-doped graphitic carbon nanotubes-on-nanosheets prepared by coordination-induced pyrolysis for oxygen reduction reaction and rechargeable Zn-air battery. J Colloid Interf Sci 639:424–433

    Article  CAS  Google Scholar 

  33. Wu DH, Huang H, Ha MU, Zhang L, Feng JJ, Wang AJ (2023) Lignin-derived iron carbide/Mn, N, S-codoped carbon nanotubes as a high-efficiency catalyst for synergistically enhanced oxygen reduction reaction and rechargeable zinc-air battery. J Colloid Interf Sci 647:1–11

    Article  CAS  Google Scholar 

  34. Guo YJ, Deng L, Li J, Guo SJ, Wang EK, Dong SJ (2011) Hemin−graphene hybrid nanosheets with intrinsic peroxidase-like activity for label-free colorimetric detection of single-nucleotide polymorphism. ACS Nano 5:1282–1290

    Article  CAS  PubMed  Google Scholar 

  35. Zhao XC (2011) Self-assembly of DNA segments on graphene and carbon nanotube arrays in aqueous solution: a molecular simulation study. J Phys Chem C 115:6181–6189

    Article  CAS  Google Scholar 

  36. Tang LH, Chang HX, Liu Y, Li JH (2012) Duplex DNA/graphene oxide biointerface: from fundamental understanding to specific enzymatic effects. Adv Funct Mater 2230:83–88

    Google Scholar 

  37. Chang F, Huang LJ, Guo CZ, Xie GM, Li JQ, Diao QZ (2019) Graphdiyne-based one-step DNA fluorescent sensing platform for the detection of mycobacterium tuberculosis and its drug-resistant genes. ACS Appl Mater Interfaces 11:35622–35629

    Article  CAS  PubMed  Google Scholar 

  38. Liu BW, Sun ZY, Zhang X, Liu JW (2013) Mechanisms of DNA sensing on graphene oxide. Anal Chem 85:7987–7993

    Article  CAS  PubMed  Google Scholar 

  39. Li SY, Cheng Q, Wang JX, Li XW, Zhang ZY (2018) CRISPR-Cas12a-assisted nucleic acid detection. Cell Discovery 4:20

    Article  PubMed  PubMed Central  Google Scholar 

  40. Shi K, Xie SY, Tian RY, Wang S, Lu Q, Gao DH, Lei CY, Zhu HZ, Nie Z (2021) A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci Adv 7:7802

    Article  Google Scholar 

Download references

Funding

This research was supported by the Science and Technology Innovation Program of Hunan Province (2021RC5028), Hunan Provincial Natural Science Foundation of China (2023JJ50229), Scientific Research Fund of Hunan Provincial Education Department (21A0295), and the Open Fund of Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education of China (E22207, E22333 E22304).

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Authors and Affiliations

  1. Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Hunan University of Science and Technology, Xiangtan, 411201, People’s Republic of China

    Yu Jiang, Xinmei Qian, Mingyu Zheng, Keqin Deng & Chunxiang Li

  2. Hunan Province College Key Laboratory of Molecular Design and Green Chemistry, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, 411201, China

    Yu Jiang, Xinmei Qian & Chunxiang Li

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  1. Yu Jiang
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  2. Xinmei Qian
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Correspondence to Keqin Deng or Chunxiang Li.

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Jiang, Y., Qian, X., Zheng, M. et al. Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors. Microchim Acta 191, 43 (2024). https://doi.org/10.1007/s00604-023-06123-0

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