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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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Cas9 protein accumulation and its catalytic activity with decreasing concentrations of pHcoCas9. (a) BsgI restriction digest assay of transgenic 16c plants co-infiltrated with cultures of TRBO-G-3′gGFP at OD₆₀₀ 0.5 and pHcoCas9 using OD₆₀₀ values of 0.5, 0.25, 0.125, and 0.0625. Agroinfiltrated N. benthamiana leaf tissue was sampled at 7 dpi. The red arrow indicates indel containing DNA. (b,c) Cas9 (164-kDa) expression was analyzed at 7 dpi using western blotting of infiltrated plants using concentrations of 0.5, 0.25, 0.125, and 0.0625 gGFP delivery constructs in the top panels. Protein loading controls were assayed through Coomassie Blue protein staining and visualization of Rubisco (55-kDa). (b) pHcoCas9 co-infiltrated with TRBO-G-3′gGFP, a TMV replicase-producing construct. (c) pHcoCas9 co-infiltrated with the TRBO replicase mutant (RM) construct RM-gGFP.