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. 2017 Nov 22;8:1688. doi: 10.1038/s41467-017-01695-x

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© The Author(s) 2017

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. 4 — CRISPR-AID for combinatorial metabolic engineering. a Yeast surface display of recombinant proteins as a representative example of combinatorial metabolic engineering. Protein folding and secretory machinery, protein super-glycosylation and other surface-displayed proteins, and degradation pathways were chosen as the targets for CRISPRa, CRISPRi, and CRISPRd, respectively. b Combinatorial optimization of EGII display on the yeast surface. EGII activities of the FACS enriched optimal combination (AID-FACS16) and those with the corresponding single component (A-pSg221, I-pSg230, and D-pSg205) were measured. c Verification of CRISPRa (PDI1) and CRISPRi (MNN9) for transcriptional regulation using qPCR. d Exploration of the synergistic interactions among activated (PDI1), interfered (MNN9), and deleted (PMR1) metabolic engineering targets. EGII activities of the double mutants, including AI-pSg417, AD-pSg418, and ID-pSg419, were measured. e Single-factor optimization of EGII display on the yeast surface. EGII activities of the strains with one gRNA (A-pSg218, I-pSg204, and D-pSg186) and the combination of the ones with the highest activities in each category (AID-pSg257) were measured. Error bars represent the mean ± s.d. of biological triplicates