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. 2015 Jul 7;14:97. doi: 10.1186/s12934-015-0288-3

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© Ronda et al. 2015

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 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.

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Multiple simultaneous integration of three β-carotene pathway genes. a Picture of colonies after simultaneous integration of BTS1, crtYB and crtI on plates without selection. S. cerevisiae (TC-3) harboring a centromeric plasmid constitutively expressing cas9 was co-transformed with: left empty vector control and linearized donor DNAs encoding BTS1, crtYB and crtI. Right gRNA vector expressing three gRNAs targeting intergenic loci XII-5, XI-2 and X-3, and linearized donor DNAs encoding BTS1, crtYB and crtI. Colonies with successful pathway integration accumulate β-carotene, resulting in an orange pigmentation. b Percentage of complete pathway integration with and without the expression of targeting gRNAs. c Frequency of correct simultaneous integration of the three genes BTS1, crtYB, and crtI at the specific intergenic loci XII-5, XI-2 and X-3, respectively, determined by genotyping. d HPLC analysis of β-carotene production of three independent orange colonies and a non-producing strain as control (CEN.PK113-7D). The experiment was repeated twice and error bars in all panels represent 95% confidence intervals.