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. 2021 Oct 22;11:20888. doi: 10.1038/s41598-021-00006-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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The preparation of cas12a condition for species authentication. (A) gRNA-A and gRNA-B design based on the multiple alignment of trnL loci and the construction of double stranded DNA as temples for gRNA synthesis. (B) The synthesis of gRNA-A and gRNA-B by in vitro transcription with T7 RNA polymerase and the synthesized gRNAs were detected by agarose gel electrophoresis. (C) The schematic illustrates the mechanism of cas12a to form binary and tertiary complex to cleave the reporter single stranded DNA as result of the fluorescence signal. (D) The condition optimization of in vitro digestion of cas12a by varying the concentration ratio of cas12a and gRNA at 1: 1 to produce the fluorescence at 37 °C for an hour. Phyllanthus species were studied including Phyllanthus amarus (PA), Phyllanthus urinaria (PU), Phyllanthus debilis (PD), Phyllanthus virgatus (PV). Initial image of agarose gel electrophoresis is shown in Figure S1.