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Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification

文献类型： 外文期刊

作者： Yao, Zhihao 1 ; He, Kaiyu 4 ; Wang, Hongmei 4 ; Feng, Suyin 6 ; Ding, Xiaoqing 7 ; Xu, Yan 2 ; Wang, Qiang 4 ; Xu, Xiahong 4 ; Wu, Qun 1 ; Wang, Liu 4 ;

作者机构： 1.Jiangnan Univ, Key Lab Ind Biotechnol, Lab Brewing Microbiol & Appl Enzymol, Minist Educ, Wuxi 214122, Jiangsu, Peoples R China

2.Jiangnan Univ, State Key Lab Food Sci & Technol, Wuxi 214122, Jiangsu, Peoples R China

3.Jiangnan Univ, Sch Biotechnol, Wuxi 214122, Jiangsu, Peoples R China

4.Zhejiang Acad Agr Sci, State Key Lab Managing Biot & Chem Threats Qual &, Hangzhou 310021, Peoples R China

5.Zhejiang Acad Agr Sci, Inst Agroprod Safety & Nutr, Hangzhou 310021, Peoples R China

6.Jiangnan Univ, Affiliated Hosp, Dept Neurosurg, Wuxi 214122, Peoples R China

7.Jiangnan Univ, Affiliated Hosp, Dept Lab Med, Wuxi 214062, Peoples R China

8.Minist Agr & Rural Affairs, Key Lab Informat Traceabil Agr Prod, Hangzhou 310021, Peoples R China

关键词： quantification; CRISPR/Cas12a; isothermal amplification; nucleic acid; one pot

期刊名称：ACS SENSORS （ 影响因子：8.9； 五年影响因子：9.0 ）

ISSN： 2379-3694

年卷期： 2024 年

页码：

收录情况： SCI

摘要： Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

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