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. 2022 Jan 25;8:819681. doi: 10.3389/fvets.2021.819681

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Copyright © 2022 Li, Lin, Feng, Lin, Guan, Chen, Liang, Wang, Li, You and Chen.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Development of the CRISPR/Cas12a assay for detection of AHPND virulence genes Pir^VPA and Pir^VPB. (A) Schematic diagram of the CRISPR/Cas12a. (B) Cleavage activity of crRNA-guided Cas12a. M: DL 2000 DNA marker; 1: intact dsDNA template (1794 bp); 2-5: The cleavage products of dsDNA template induced by crRNA1, crRNA2, crRNA3, crRNA4; 6: The cleavage products of dsDNA template induced by mixture of crRNA1 to crRNA4. The expected cleavage products are marked by red arrows. (C) Validation of collateral cleavage activity of Cas12a by fluorescence assay. The DNA template in positive group was plasmid pUC19-PirAB, and negative group was blank vector pUC19, while reporter only group was reaction without DNA template. (D) Comparison of different crRNAs targeting Pir^VPA and Pir^VPB by fluorescence assay. (E) Heatmap showing the optimal concentrations of Cas12a and crRNA based on fluorescence intensity. Each row presents the concentration of Cas12a used, each column presents the concentration of crRNA used. Error bars represent the standard deviation from three independent experiments. ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 and ns, not significant.