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. 2024 Aug 13;20:123. doi: 10.1186/s13007-024-01234-y

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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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Cas9 and Cas12a toolkit parts available via AddGene. Plasmid parts of the toolkit: Level 1 guide accepters for Cas9 and Cas12a with three promoter options. Compatible binary vectors containing plant selection markers and nuclease cassettes which are wheat or barley specific. P-35S = Cauliflower mosaic virus long 35S promoter, P-PvUbi = Panicum virgatum ubiquitin promoter, HptII = hygromycin phosphotransferase CDS with intron, T-35S = Cauliflower mosaic virus terminator, P-ZmUbi = Zea mays ubiquitin promoter, ttAtCas12a + 8int = temperature tolerant Arabidopsis codon optimised Cas12a CDS with 8 introns, ZmCas9 + 13int = Zea mays codon optimised Cas9 with thirteen introns, T-Nos = nopaline synthase terminator, GRF-GIF = Growth-regulating factor 4 (GRF4) and its cofactor GRF interacting factor 1(GRF1) fusion, GR-GRF-GIF = Rat glucocorticoid receptor (GR) & Growth-regulating factor 4 (GRF4) & its cofactor GRF interacting factor 1(GRF1) fusion. RFP = red fluorescent protein for colour selection during cloning, TaU6 = Triticum aestivum U6 promoter, TaU3 = Triticum aestivum U3 promoter, HvU3 = Hordeum vulgare U3 promoter, HH = Hammerhead ribozyme, HDV = hepatitis delta virus ribozyme, tRNA = tRNA sequence, DR = short invariable 5’ region of Cas12a guide, sgRNA = invariable 3’ region of Cas9 guide, LacZ = blue/white colour selection marker for cloning, BsaI = restriction site, BpiI = restriction site. Triangles indicate GoldenGate cloning site cut points. Arrows indicate direction of transcription