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Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

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  • Jeong Moon

    (University of Connecticut Health Center)

  • Changchun Liu

    (University of Connecticut Health Center)

Abstract

Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans-cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings.

Suggested Citation

  • Jeong Moon & Changchun Liu, 2023. "Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-43389-7
    DOI: 10.1038/s41467-023-43389-7
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    1. Xiong Ding & Kun Yin & Ziyue Li & Rajesh V. Lalla & Enrique Ballesteros & Maroun M. Sfeir & Changchun Liu, 2020. "Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    2. Kean Hean Ooi & Mengying Mandy Liu & Jie Wen Douglas Tay & Seok Yee Teo & Pornchai Kaewsapsak & Shengyang Jin & Chun Kiat Lee & Jingwen Hou & Sebastian Maurer-Stroh & Weisi Lin & Benedict Yan & Gabrie, 2021. "An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing," Nature Communications, Nature, vol. 12(1), pages 1-23, December.
    3. Margot Karlikow & Evan Amalfitano & Xiaolong Yang & Jennifer Doucet & Abigail Chapman & Peivand Sadat Mousavi & Paige Homme & Polina Sutyrina & Winston Chan & Sofia Lemak & Alexander F. Yakunin & Adam, 2023. "CRISPR-induced DNA reorganization for multiplexed nucleic acid detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    4. Mu-Sen Liu & Shanzhong Gong & Helen-Hong Yu & Kyungseok Jung & Kenneth A. Johnson & David W. Taylor, 2020. "Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
    5. Eman A. Ageely & Ramadevi Chilamkurthy & Sunit Jana & Leonora Abdullahu & Daniel O’Reilly & Philip J. Jensik & Masad J. Damha & Keith T. Gagnon, 2021. "Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
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