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. 2021 Mar 24;11:6769. doi: 10.1038/s41598-021-85366-4

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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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TRBO-HcoCas9-delivered Cas9 expression and catalytic activity with the RNA silencing suppressor P19. (a) Schematic representation of the Cas9 delivery vector TRBO-HcoCas9. (b) TRBO-HcoCas9-delivered Cas9 protein expression co-infiltrated with and without a P19-expressing plasmid in N. benthamiana from 3–9 dpi. The top panels for each day are western blots used to detect Cas9 protein (164-kDa). The bottom panels are protein gels stained with Coomassie to detect Rubisco (55-kDa), the loading control. (c) BsgI restriction assay of 4-week-old 16c plants co-infiltrated with TRBO-HcoCas9/TRBO-G-3′gGFP sampled at 10 dpi, with and without P19, to analyze functionality of the delivered Cas9 protein. The increased Cas9 expression (b) reflects the enhanced indel induction (c; red arrow) in the presence of P19. The average and standard deviation of indel percentages were calculated based on three independent biological replicates.