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. 2022 Nov 26;5:1296. doi: 10.1038/s42003-022-04258-z

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Schematic showing the pmTmG reporter assay for comparing different PAMs of Nme1Cas9, Nme2Cas9, and NcCas9 orthologs. Variable PAM sequences were introduced into the constructs. In the presence of a functional PAM, cleavage-mediated tdTomato cassette excision was performed, which led to the expression of EGFP, thereby allowing live visualization under a fluorescence microscope or detection via flow cytometry analysis (FCA). b Comparison of Nme1Cas9, Nme2Cas9, and NcCas9 cleavage efficiency of different PAMs by FCA. The different PAMs were designed based on the optimal GTACGTAT PAM sequence. Red or green represents the tdTomato or EGFP signal, respectively. c Representative fluorescence microscopy images of PAM analysis. Scale bar, 200 μm. Error bars indicate the s.e.m. (n = 3).