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CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity

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Abstract

Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of  a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3’ overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5’ phosphate termini and exposing a single-strand region allowing complementary to the 3’ overhang. After hybridization, the 3’ and 5’ termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.

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Funding

This work was supported by Hong Kong Research Grants Council (11217820 and N_CityU119/19); the Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20210324134006017); and City University of Hong Kong (9678242, 6430620, and 7020072).

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Authors and Affiliations

  1. Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong, Special Administrative Region, 999077, Hong Kong, China

    Chenyu Cui & Ting-Hsuan Chen

  2. City University of Hong Kong Shenzhen Research Institute, Shenzhen, 518000, China

    Ting-Hsuan Chen

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  1. Chenyu Cui
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  2. Ting-Hsuan Chen
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C.C.: conceptualization, methodology, investigation, data curation, and writing, original draft preparation. T.-H.C.: conceptualization, methodology, supervision, funding acquisition, and writing reviewing and editing.

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Correspondence to Ting-Hsuan Chen.

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Cui, C., Chen, TH. CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity. Microchim Acta 190, 468 (2023). https://doi.org/10.1007/s00604-023-06050-0

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