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Fast and sensitive CRISPR detection by minimized interference of target amplification

Nature Chemical Biology (2024)Cite this article

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Abstract

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10–10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp μl−1, making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp μl−1. The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection.

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Fig. 1: Mutants of AapCas12b enable faster and more sensitive one-pot testing than WT.
Fig. 2: Detection of SARS-CoV-2 clinical samples using the AapCas12b mutant.
Fig. 3: REVERSE enables faster and more sensitive one-pot testing than SHINE.
Fig. 4: REVERSE-2 enables nucleic acids detection at room temperature.

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Acknowledgements

This work is kindly supported by Key R&D Program of Hubei Province (grant nos. 2022BCA089 to H.Y. and 2022ACA005 to Y.Z.), National Key R&D Program of China (grant nos. 2019YFA0802801, 2018YFA0801401 and 2022YFF1002801), the Ministry of Agriculture and Rural Affairs of China, the Strategic Priority Research Program of CAS (grant no. XDB29010300 to X.Z.), the National Natural Science Foundation of China (grant nos. 31871345 and 32071442 to H.Y., 31972936 to Y.Z. and 31970169 to X.Z.), State Key Laboratory for Animal Disease Control and Prevention (SKLVBF202202), Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (grant no. TFJC2018004), the Fundamental Research Funds for the Central Universities (grant nos. 2042022dx0003 and 2042022kf1190), Applied Basic Frontier Program of Wuhan City (grant no. 2020020601012216 to H.Y.) and the startup funding from Wuhan University (to H.Y. and Y.Z.). We thank the core facility of Medical Research Institute at Wuhan University for their technical supports. We thank Wuhan Easy Diagnosis Biomedicine Co. for providing some reagents.

Author information

Author notes

  1. These authors contributed equally: Xiaohan Tong, Kun Zhang, Yang Han.

Authors and Affiliations

  1. Department of Clinical Laboratory, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Xiaohan Tong, Kun Zhang, Tianle Li, Min Duan, Ruijin Ji, Ying Zhang & Hao Yin

  2. State Key Laboratory of Virology, TaiKang Centre for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China

    Xiaohan Tong, Kun Zhang, Tianle Li, Min Duan, Ruijin Ji & Hao Yin

  3. Center for Translational Medicine, Wuhan Jinyintan Hospital, Wuhan, China

    Yang Han & Xianguang Wang

  4. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

    Xi Zhou

  5. Department of Rheumatology and Immunology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Ying Zhang

  6. Department of Urology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Hao Yin

  7. Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, China

    Hao Yin

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Contributions

H.Y. conceived, designed and managed the project. X.T. and K.Z. performed most experiments with the help of T.L., M.D. and R.J. X.T. engineered the Cas12b protein and developed a room temperature REVERSE method. X.T., K.Z. and T.L. developed one-pot testing mediated by Cas12b-mutants. X.T., K.Z. and T.L. optimized conditions for detecting viral samples. Y.Z. provided conceptual advice. X.Z., X.W. and Y.H. provided samples, and Y.H. performed related experiments. X.T. and H.Y. analyzed the data. H.Y., Y.Z. and X.T. wrote the paper with inputs from the authors.

Corresponding author

Correspondence to Hao Yin.

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Competing interests

H.Y., Y.Z., X.T., K.Z. and T.L. have filed a patent application (no. 202311783425.X) through Wuhan University. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Comparison of Cas12b-mediated one-pot testing using canonical PAM or suboptimal PAMs.

(a–f) The fluorescence curves of one-pot testing using canonical PAM or suboptimal PAMs of Cas12b. For a-c, the one-pot testing was mediated by LAMP. For d-f, the one-pot testing was mediated by RPA. Mean ± s.d. for 3 technical replicates.

Source data

Extended Data Fig. 2 Sequences of AapCas12b.

Conservative analysis of residues 478 and 396 between Cas12b orthologs.

Extended Data Fig. 3 Cis-cleavage of Cas12b variants.

Cis-cleavage of Cas12b-WT, Cas12b-2M, enzymatically dead Cas12b-WT, and enzymatically dead Cas12b-2M. Enzymatically dead indicates E848A (ref. 45). Proteinase K was introduced to stop reaction at various time points. The experiment was repeated twice with similar results. S, 1241 bp substrate; P1 and P2, 927 bp product 1 and 314 bp product 2.

Source data

Extended Data Fig. 4 Optimization of one-pot testing.

(a) The performance of commercially available bst enzymes were determined in one-pot testing at 60 °C. Bst1-7 represent bst 2.0 DNA polymerase (NEB), bst 3.0 DNA polymerase (NEB), bst DNA polymerase from Vazyme, Beyotime, FAPON-BIOTECH, YEASEN and bst DNA polymerase Plus from YEASEN. (b) The optimized one-pot testing using Cas12b-2M or WT. ‘cp/µL’ indicates the copy number per microliter in one-pot testing. For a & b, mean ± s.d. for 3 technical replicates. (c) LAMP alone tests of SARS-CoV-2 clinical samples.

Source data

Extended Data Fig. 5 The fluorescence curves in one-pot testing with or without UNG.

(a-b) The fluorescence curves of one-pot testing with or without UNG were identified. The Ct values of clinical samples 1-6 were 23.8, 23.5, 22.6, 27.0, 33.5, and 29.0, respectively. Mean ± s.d. of n = 3 technical replicates.

Source data

Extended Data Fig. 6 Performance of REVERSE in detecting SARS-CoV-2.

(a) Sensitivity comparison between REVERSE and SHINE. The black dotted line represents the threshold, which was determined as three times the negative control fluorescence value. (b, c) Evaluation of REVERSE-2 performance at 20 °C using a fluorescence reader (b) and blue light (c). (d) Comparison of Cas13a-, Cas12a- and Cas12b-mediated one-pot testing at 20 °C and 25 °C. The input substrates comprised pseudo-SARS-CoV-2 RNA of 1 cp/μL. Results were captured using blue light at 30, 40, 50, and 60 minutes. Mean ± s.d. for 4 technical replicates for a; 3 technical replicates for b.

Source data

Extended Data Fig. 7 Sensitivity and specificity comparison between REVERSE-2 and antigen test at room temperature.

(a, b) Sensitivity assessment of REVERSE-2 (a) and the antigen test (b). (c, d) Determination of the specificity of REVERSE-2 and the antigen test.

Source data

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Tong, X., Zhang, K., Han, Y. et al. Fast and sensitive CRISPR detection by minimized interference of target amplification. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01534-9

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