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. 2019 Oct 18;2:383. doi: 10.1038/s42003-019-0637-6

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© The Author(s) 2019

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. 3 — dCas9 and type I-E Cascade chromosomal gene activation in Zea mays. a Strategy for activating the Zea mays anthocyanin pigment pathway in Hi-Type II immature embryos. C1 and R transcription factors complex to activate the Bronze 1 (Bz1) gene resulting in anthocyanin production. By overexpressing the C1 coding sequence (CDS) using a Cauliflower Mosaic Virus (CMV) enhancer (35S) and promoter (Pro), RNA-guided transcriptional activation of the chromosomal r gene results in an anthocyanin pelargonidin (red) cellular phenotype. b Negative and positive controls for r gene based activation of anthocyanin. The negative control (-crRNA Ctrl) comprised transformations performed with SthCascade expression cassettes (CasA-CBF1, CasB, CasC, CasD-CBF1, CasE-CBF1) and the C1 CDS transgenic over-expression construct in the absence of a crRNA transcriptional cassette. Co-delivery of C1 and R transgenic over-expression cassettes served as a positive control. c SthCascade (CasA-CBF1, CasB, CasC, CasD-CBF1, and CasE-CBF1) and dCas9-CBF1 anthocyanin induction 48 h after transformation for each of the corresponding SthCascade cRNAs or dCas9 sgRNAs. d SthCascade (CasA-CBF1, CasB, CasC, CasD-CBF1, and CasE-CBF1) and dCas9-CBF1 anthocyanin phenotype when all 3 SthCascade crRNAs or dCas9 sgRNAs were co-delivered. Photos were taken 48 h post-transformation. e Quantification of anthocyanin phenotype resulting from SthCascade and dCas9 transcriptional activation. Anthocyanin positive cells were counted on the surface of three independent microprojectile transformations. Treatments were not significantly different based on a one-sided t-test (p = 0.278)