Wicket: a versatile tool for the integration and optimization of

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Wicket: a versatile tool for the integration and optimization of exogenous pathways in Saccharomyces cerevisiae Sha Hou, Qin Qin, and Junbiao Dai ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00391 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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ACS Synthetic Biology

Wicket: a versatile tool for the integration and optimization of exogenous pathways in Saccharomyces cerevisiae

Sha Hou1,†, Qin Qin1,†, Junbiao Dai1,2,* 1

Key Laboratory of Industrial Biocatalysis (Ministry of Education) and Center for

Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China 2

Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes

of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

†

These authors contributed equally to this work

*

Corresponding author. E-mail: [email protected]

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Abstract: Yeast can be used as a microbial cell factory to produce valuable chemicals. However, introducing an exogenous pathway into particular or different chromosomal locations for stable expression is still a daunting task. To address this issue, we designed a DNA cassette called a “wicket”, which can be integrated into the yeast genome at designated loci to accept exogenous DNA upon excision by a nuclease. Using this system, we demonstrated that, in strains with “wickets”, we could achieve near 100% efficiency for integration of the β-carotene pathway with no need for selective markers. Furthermore, it allowed independent and simultaneous integration of different genes in a pathway, resulting in a large variety of strains with variable copy numbers of each gene. This system could be a useful tool to modulate the integration of multiple copies of genes within a metabolic

pathway and to optimize the yield of the target products. Keywords: Saccharomyces cerevisiae, CRISPR/Cas, multi-copy integration, tandem duplication, cocktail integration, pathway optimization

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Recently, microorganisms have increasingly been used to help solve global issues such as environmental contamination, health problems, and energy crises, for at least the following three reasons: I) a large variety of microorganisms exist;1,2 II) microorganisms often have a compact genome and relatively clear genetic background, which allows easy and convenient genetic manipulation to repurpose them for specific demands; and III) the majority of microorganisms divide rapidly and can accumulate a large amount of biomass within a short time period, making them ideal for the production of value-added products. One of the most promising microorganisms is the budding yeast, Saccharomyces cerevisiae, which has for years served as a good microbial cell factory for various highly valued products, such as fine chemicals and pharmaceuticals.3–5 In metabolic engineering, reconstruction of a given pathway in a MCF is usually the first step, followed by numerous optimization procedures. Although there are a variety of tools to manipulate the yeast genome, generating a highly producing strain is still a rate-limiting step. Much time and effort has been put into developing more convenient and efficient strain engineering methods, with three major attempts having been made. First, rapid reconstruction of heterologous metabolic pathways has been attempted by expressing all the required genes from plasmids. However, the consistency among cells in a population and the stability of plasmids during cell division are two questions that should be considered in this 3



approach.6,7 Alternatively, all genes in a pathway could be integrated into the host genome, allowing them to be stably inherited. Unfortunately, the application of this approach has been limited by the low efficiency of integration and the labor-intensive cloning procedure for large DNA fragments. Previous studies have devoted a lot of effort to exploring cloning methods and increasing integration efficiency.8–10 The second attempt is optimization of exogenous pathways. For example, the expression level of each gene is fine-tuned through promoter screening to balance the amount of each enzyme in the pathway for the best coordination.11,12 On the other hand, some groups have taken advantage of the presence of native repetitive sequences in the yeast genome, such as the retrotransposon Ty1 or ribosome sequence, for the integration of multiple copies of genes in the pathway for overexpression.13,14 The third attempt is engineering of chassis cells or redirection of metabolic fluxes to increase the yield of products by increasing substrate and decreasing by-product levels.15 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) are powerful tools for efficient DNA editing. A single guide RNA (sgRNA) is required for site-specific endonuclease activity, making marker-free multi-locus integration possible.16,17 DiCarlo et al.18 first achieved highly efficient DNA editing using the CRISPR/Cas9 system in S. cerevisiae in 2013. Soon after, CRISPR/Cas9 was used for multiplex genome 4


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editing with up to five sites simultaneously and achieved relatively high efficiency.6,7,19–21 All these studies have contributed greatly to the development of genome editing in Saccharomyces cerevisiae. High targeting efficiency of CRISPR/Cas9 in budding yeast provides a new solution for strain engineering, making multi-locus, marker-free exogenous pathway integration possible. Recently, Shuobo Shi used CRISPR/Cas9 to target the δ-element,14 a direct repeat from the yeast retrotransposons, for integration of metabolic pathways, dramatically increasing the efficiency and allowing insertion of multiple copies of the same set of genes with no requirement for selective markers. However, retrotransposons are unstable regions in the yeast genome, and strict genetic stability detection of engineered strains is necessary when using this method. Therefore, a convenient and stable integration method for exogenous pathways is urgently required for widespread use of yeast cell factories. To facilitate the integration of exogenous genes into the yeast genome, we designed a short DNA cassette composed of two 50 bp DNA fragments (called universal left and right homology arms, or uni-HAL and uni-HAR for short) with no homology in the yeast genome and a 23 bp CRISPR/Cas9 target sequence, consisting of a 20 bp guide RNA (gRNA) and a 3 bp protospacer adjacent motif (PAM), in the middle (Figure 1 and Figure S1). With the help of CRISPR/Cas9, these “wickets” could be inserted into the yeast genome with multi-copies, and 5



served as docks for exogenous pathways. The 23 bp target sequence of the “wicket” was recognized and excised by the Cas9/sgRNA complex with high efficiency to generate a double-strand break (DSB), allowing integration of exogenous genes with uni-HAL and uni-HAR, which were used as donors when the yeast cell performed DSB repair through the homologous recombination pathway. According to the forms of exogenous pathways to be integrated into the yeast genome, either a pre-assembled pathway or a cocktail of single genes could be used. Consequently, this DNA cassette is just like the “gate” of chromatin, in which the two homology arms serve as two doors and the Cas9 recognition site is like a lock. Accordingly, a specific gRNA guides Cas9 to the “wicket” locus as a key to “unlock” the “door” and the exogenous genes seal the door after DSB repair through homologous recombination (Figure 1). To install the “wicket” into the genome, a strain with pTDH3-Cas9 integrated into the genome (yQ9) was used, allowing stable and constitutive expression of Cas9 (Table S1). To facilitate high efficiency targeting, the SNR52 promoter and SUP4 terminator were used for the expression of sgRNA (Figure S2). We first tested multiplex targeting efficiency at three marker gene loci, ADE2, CAN1, and URA3, using two different strategies. The first was to co-transform the strain with multiple plasmids containing different sgRNAs (Figure 2a, I), which resulted in very low co-targeting efficiency. The other strategy was to transform the yeast with three 6


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sgRNA fragments plus a linear vector, which shared about 60 bp overlap, creating three complete plasmids via homologous recombination in the yeast cell (Figure 2a, II). This method resulted in nearly 100% co-targeting efficiency at the ADE2, CAN1, and URA3 genes simultaneously (Figure 2a, II). Therefore, this gap repaired sgRNA method was adopted in all future experiments. Next, we tested insertions of the “wicket” into different numbers of intergenic regions. When targeting three sites, we found that five and two out of the eight colonies contained the “wickets” integrated at all three designated locations in two independent attempts (Figure 2b). The co-targeting efficiency was a little lower than when targeting the three marker genes (Figure 2a) but high enough to obtain a candidate clone easily. Furthermore, we also tested if we could insert the “wicket” at four locations simultaneously. In two attempts with completely different sets of sgRNAs, we obtained four clones and one clone with “wickets” correctly integrated into all four locations (Figure 2b). With this method, we sequentially inserted three “wickets” at a time using LEU2 and URA3 selection markers alternately, and successfully constructed a series of strains containing either 3 (SHY123), 6 (SHY124), 9 (SHY125), or 12 (SHY126) “wickets” (Figure S3 and Table S1). All the gRNAs are listed in Table S4. To demonstrate the application of “wickets” for integrating multiple copies of an exogenous metabolic pathway, we used the β-carotene synthesis pathway as an 7



example, which requires the presence of three additional genes, CrtE, CrtI, and CrtYB, to enable the yeast to produce β-carotene.11,22–24 We first constructed a plasmid (pSH200) with the five parts, uni-HAL, pZD90-CrtYB, pZD93-CrtI, pZD96-CrtE, and uni-HAR, adjacent to each other, which could be released as a single fragment when treated with restriction enzyme BssHII. Since all genes in the pathway were cloned together before inserting into the genome, we named this strategy pre-assembled integration. The digested pSH200 was co-transformed into the “wicket”-containing yeast strains with pSH201, the plasmid containing the sgRNA targeted to the “wicket”. Production of β-carotene made the yeast colony orange, enabling easy identification of colonies with the correct integration. Strain SHY123 presented almost 100% integration efficiency, and strains SHY124 and SHY125 showed about 50% efficiency, whereas the integration efficiency of SHY126 was very low (Figure 3a). Presumably, more accepting sites would give more integration opportunity; however, the integration efficiency of these four “wicket” strains argues against this assumption. One potential explanation is that, in strains with a lot of “wickets”, Cas9 generated too many DSBs in the genome and the DNA repair machinery could not repair all of them. Alternatively, it is possible that the integration of many exogenous genes produced too much metabolic burden, reducing cell survival. We prefer the first hypothesis since the insertion of non-coding DNA fragments showed the same low 8


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efficiency (see Figure S4) and white colonies possessed an intact “wicket” at all the loci, indicating lower Cas9 excision efficiency in these viable colonies (Figure S5). Nevertheless, the integration efficiency in SHY123, SHY124, and SHY125 should be sufficient for the introduction of an exogenous pathway. To estimate the copy number of integrated exogenous pathways, eight colonies were randomly isolated from transformed SHY123 and subjected to qPCR. Surprisingly, all of them possessed more than three copies of exogenous genes, and one colony even had more than 20 copies (Figure 3b). Since SHY123 contained only three “wickets”, there must be some mechanisms leading to increased copy numbers in these colonies. One possibility is that the repair of the DSB resulted in tandem duplication of the integrated DNA. To test this hypothesis, we designed a pair of primers near the ends of the integrated DNA pointing outwards (Figure 3c). Only when two or more copies of the DNA fragments were positioned adjacent to each other could a DNA fragment be amplified. This analysis suggested the presence of tandem duplication, consistent with previous studies, in which “tandem duplication” was identified when the researchers tried to integrate multiple copies of genes within Ty elements.9,25 The presence of duplicated fragments was further confirmed by DNA sequencing (Figure 3d). Of six duplication fragments, four seemed to ligate through BssHII sticky ends, which indicated that tandem duplication preceded integration. The remaining two were obviously formed 9



by more complicated recombination events because of the absence of uni-HAL, uni-HAR, and BssHII recognition sites. Furthermore, we evaluated the stability of strains containing high copy numbers of β-carotene genes and found that they could be maintained for more than 100 generations under non-selective conditions (Figure 3b). Although we used the β-carotene synthesis pathway as an example to demonstrate the power of the “wicket” because it is easy to identify clones with high production by examining their color, “wickets” can be applied to any metabolic pathway to generate a pool of strains with various amounts of desired products. However, an efficient selection or screening method will be required. Therefore, the “wicket” provides a simple method to quickly integrate multiple copies of genes in a pathway into the yeast genome, which allows overexpression of several genes simultaneously to boost the productivity of the target metabolites. The pre-assembled integration allows overexpression of all genes within a pathway, which in most circumstances might be necessary. However, sometimes only a few key genes should be highly expressed whereas the expression of the remaining genes in the pathway must be tightly controlled to maximize the metabolic flux. Therefore, we designed an alternative strategy named cocktail integration, which was first applied for the integration of xylose metabolic genes into retrotransposon δ-sites within the yeast genome.26,27 In this method, every gene 10


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with its promoter and terminator was PCR-amplified to incorporate the flanking uni-HAL and uni-HAR. Since, during transformation, we selected the sgRNA plasmid using an auxotrophic marker, there was no need to select the integrated genes using an additional marker, making “wicket” a marker-free system. Each gene was integrated into the “wickets” independently, leading to various copy numbers. Again, using the β-carotene synthesis pathway as an example, we first tested if cocktail integration could efficiently produce β-carotene by incorporating all required genes within the pathway in a yeast strain. The production of orange colonies varied among different strains. SHY123 and SHY124 generated about 20% and 50% orange colonies, respectively, whereas SHY125 and SHY126 almost failed to produce any (Figure 4a). This result is promising considering that, for the cells to produce β-carotene, at least one copy of each of the three genes has to be integrated. Next, we tested if cocktail integration could produce strains with a wide range of β-carotene production. Eight colonies were randomly isolated from SHY124 transformed with the PCR mixture. The eight clones produced different amounts of β-carotene, with four of them producing much higher levels than the others (Figure 4b). We analyzed the copy number of each gene in these clones by qPCR. As expected, there were different numbers of CrtE, CrtI, and CrtYB genes within each 11



colony and the number varied between different colonies (Figure 4c). Intriguingly, all four colonies with a higher carotenoid yield contained many copies of CrtI and CrtYB but only a few copies of CrtE, which suggests that, to ensure the high yield of β-carotene, both CrtI and CrtYB should be highly expressed and the expression of CrtE must be kept low, consistent with our previous study.11 Therefore, the cocktail integration could not only quickly generate strains containing the exogenous pathway, but also produce hints to improve the yield of the desired products. However, the differences in copy number might only be one potential factor affecting β-carotene production. Other aspects such as the genomic location of the integrated genes might also influence their expression and subsequently alter β-carotene production (Figure S6). Interestingly, we also found the existence of “tandem duplication” within the integrated loci in cocktail integration (Figure 4d). To evaluate the formation of these duplications, a PCR-based assay was performed. Four types of tandem duplication, i.e., CrtYB 3’ to CrtYB 3’, CrtE 5’ to CrtYB 3’, CrtE 3’ to CrtI 5’, and CrtE 5’ to CrtI 3’, were identified in the four colonies (Figure 4d). Similarly, the mechanisms leading to these duplications are unknown but their presence increases the diversity of strains, which could potentially be very useful for metabolic engineering. In summary, we have developed a versatile platform, named “wicket”, facilitating efficient marker-free, multi-locus integration and convenient optimization 12


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for exogenous pathways through both pre-assembled and cocktail integration in S. cerevisiae. Pre-assembled integration presents high integration efficiency and high copy number pathway integration. Cocktail integration is a cloning-free, quick integration method for multi-enzymatic pathways, providing large variety for high yield strain selection. Therefore, the “wicket” method improves exogenous pathway integration and optimization methods in terms of both copy number and coordination of each gene.

Methods Strains and growth conditions. All the yeast strains used in this study are listed in Table S1. Starting strain yQ9 was derived from JDY52 described previously by inserting the pTDH3-derived Cas9 gene into the HO locus with Trp1 as a selection marker.7,11 Different numbers of “wickets” were integrated into yQ9, generating a series of strains as listed in Table S1. Yeast cells were cultured in either YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 0.32 g/L tryptophan) or synthetic medium (1.7 g/L YNB, 5 g/L ammonium sulfate, and 1 g/L amino acid powder) at 30°C with agitation at 220 rpm. Ade2, Ura3, and Can1 targeting using gap repaired gRNA. To apply the CRISPR/Cas9 system, we adopted the system developed by Mali et al.17 with the expression of gRNA (sgRNA) under the control of pSNR52. The sgRNA sequences 13



of three marker genes are listed in Table S4. sgRNA fragments were amplified using primers SHO794 and SHO795, and vector was amplified from pRS425 using SHO796 and SHO797. PCR product of each fragment (2.5 µl) was mixed and used for one transformation, and SC-Leu plates were used for selection. After 2 days, culture colonies were replicated onto SC plus 5-FOA (1 g/L), SC-Arg plus canavanine (50 mg/L), SC-Arg with both 5-FOA and canavanine, and SC-Leu plates. The single disruption efficiency of ADE2, URA3, and CAN1 was defined as the number of red colonies, that of colonies on 5-FOA plates, that of colonies on canavanine plates, divided by that of colonies on SC-LEU plates. The targeting efficiency of all three genes was defined by the number of red colonies on plates with both 5-FOA and canavanine divided by that of colonies on SC-Leu plates. The design of gRNA. All the “wickets” were inserted into intergenic regions of the yeast genome, and sgRNA was designed at http://crispr-era.stanford.edu/.28 All sgRNAs used in this study are listed in Table S4. Insertion of “wickets”. All sgRNAs were amplified using primers SHO794 and SHO795. The vector was amplified from either pRS425 or pRS426 using primers SHO796 and SHO797 (Table S1). pRS425 and pRS426 were used consecutively for continuous targeting. For one round of targeting, three sgRNA fragments and one type of vector, each at 2.5 µl, were transformed into the yeast strain. After each round of transformation, the sgRNA plasmids were removed to allow the next round 14


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of transformation. sgRNAs for each round were L35, L37, and L44 (round 1); L34, L39, and L51 (round 2); L31, L33, and L36 (round 3); and L47, L49, and L67 (round 4). Details and efficiency of strain construction are shown in Figure S3. Pre-assembled integration. pSH200 (10 µg) was digested with BssHII (NEB R0199L) and EcoRV (NEB R0195L) for 4 h. When performing pre-assembled integration, 1 µg of digested pSH200 together with 500 ng of pSH201 was transformed into “wicket” strains and cells were transferred to SC-URA medium after transformation. Every 2 days, a 100-fold dilution was performed in the same medium. After 5 days, a small volume of the culture was taken, diluted, and spread onto SC-Ura plates to estimate the integration efficiency. Since colonies with β-carotene genes appear orange, the integration efficiency was defined as the number of orange colonies divided by the total number of colonies. Cocktail integration method. Three genes of the β-carotene pathway, CrtE, CrtI, and CrtYB, were amplified using primers SHO714 and SHO715 for the addition of a homology arm. A mixture (10 µl) of these three PCR products was co-transformed with pSH201 into the yeast strains with “wickets” (Table S1). The following procedure was the same as with pre-assembled integration. Determining the copy number of β-carotene genes. Copy number was estimated by quantitative PCR (qPCR). All three genes, CrtE, CrtI, and CrtYB, were analyzed in cocktail integration, and only CrtE was used as the target gene for 15



pre-assembled integration. ACT1 was chosen as the reference gene. Primers were designed using primer3, and the primers for ACT1 were the same as those used previously.30,31 qPCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR System using SYBR Green (Genestar, A311-05). ZLY257, which contained single copies of CrtE, CrtI, CrtYB, and ACT1 on the chromosome, was used as a control and to establish the standard curve. The copy number was quantified using the ∆∆Cq method. The computational formula is as follows: Copy No. of Gene X=2-[Cq(Gene X)-Cq(ACT1)](sample)/2-[Cq(Gene X)-Cq(ACT1)](control) Quantification of carotenoid yield by HPLC. Extraction and HPLC analysis of carotenoids were performed using the method modified from a previous study.11 Cell culture and the extraction procedure were the same as in the previous study except that we took 50 µl of extraction supernatant to HPLC analysis directly, rather than drying and re-dissolving in another solvent.11

Supporting Information Lists of strains, plasmids, primers, sgRNA sequences, and additional figures are supplied as supporting information. This material is available on the ACS website. Author Information Corresponding Author

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*[email protected] Author Contributions S.H. and J.D. proposed the idea and designed experiments. S.H. and Q.Q. performed the experiments. S.H. and J.D. wrote the manuscript. Notes The authors declare no competing financial interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (31471254 and 31725002) and partially supported by Bureau of International Cooperation, Chinese Academy of Sciences (172644KYSB20170042). We thank Yikang Zhou and Jun Lin for help with HPLC analysis. References (1) Mahdavi, H., Ulrich, A. C., and Liu, Y. (2012) Metal removal from oil sands tailings pond water by indigenous micro-alga. Chemosphere 89, 350–354. (2) Pinjing, H., Liming, S., Zhiwen, Y., and Guojian, L. (2001) Removal of hydrogen sulfide and methyl mercaptan by a packed tower with immobilized micro-organism beads. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 44, 327–333. (3) Fletcher, E., Krivoruchko, A., and Nielsen, J. (2016) Industrial systems biology and its impact on synthetic biology of yeast cell factories. Biotechnol. Bioeng. 113, 1164–1170. (4) Hong, K.-K., and Nielsen, J. (2012) Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell. Mol. Life Sci. 69, 2671–2690. (5) Kim, I.-K., Roldão, A., Siewers, V., and Nielsen, J. (2012) A systems-level approach for metabolic engineering of yeast cell factories. FEMS Yeast Res. 12, 228–248. (6) Jensen, N. B., Strucko, T., Kildegaard, K. R., David, F., Maury, J., Mortensen, U.

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H., Forster, J., Nielsen, J., and Borodina, I. (2014) EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 14, 238–248. (7) Ryan, O. W., Skerker, J. M., Maurer, M. J., Li, X., Tsai, J. C., Poddar, S., Lee, M. E., DeLoache, W., Dueber, J. E., Arkin, A. P., and Cate, J. H. (2014) Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3. (8) Eckert-Boulet, N., Pedersen, M. L., Krogh, B. O., and Lisby, M. (2012) Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae. Yeast 29, 323–334. (9) Maury, J., Germann, S. M., Jacobsen, S. A. B., Jensen, N. B., Kildegaard, K. R., Herrgård, M. J., Schneider, K., Koza, A., Forster, J., Nielsen, J., and Borodina, I. (2016) EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae. PLOS ONE 11, e0150394. (10) Mitchell, L. A., Chuang, J., Agmon, N., Khunsriraksakul, C., Phillips, N. A., Cai, Y., Truong, D. M., Veerakumar, A., Wang, Y., Mayorga, M., Blomquist, P., Sadda, P., Trueheart, J., and Boeke, J. D. (2015) Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Res. 43, 6620– 6630. (11) Guo, Y., Dong, J., Zhou, T., Auxillos, J., Li, T., Zhang, W., Wang, L., Shen, Y., Luo, Y., Zheng, Y., Lin, J., Chen, G.-Q., Wu, Q., Cai, Y., and Dai, J. (2015) YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 43, e88. (12) Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J., and Dueber, J. E. (2013) Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668–10678. (13) Lopes, T. S., Klootwijk, J., Veenstra, A. E., van der Aar, P. C., van Heerikhuizen, H., Raué, H. A., and Planta, R. J. (1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79, 199–206. (14) Shi, S., Liang, Y., Zhang, M. M., Ang, E. L., and Zhao, H. (2016) A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae. Metab. Eng. 33, 19–27. (15) Zhou, P., Ye, L., Xie, W., Lv, X., and Yu, H. (2015) Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Appl. Microbiol. Biotechnol. 99, 8419–8428. (16) Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., and Zhang, F. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823. (17) Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S.,

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Yang, L., and Church, G. M. (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838. (18) DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., and Church, G. M. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343. (19) Ronda, C., Maury, J., Jakočiunas, T., Jacobsen, S. A. B., Germann, S. M., Harrison, S. J., Borodina, I., Keasling, J. D., Jensen, M. K., and Nielsen, A. T. (2015) CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microb. Cell Fact. 14, 97. (20) Walter, J. M., Chandran, S. S., and Horwitz, A. A. (2016) CRISPR-Cas-Assisted Multiplexing (CAM): Simple Same-Day Multi-Locus Engineering in Yeast. J. Cell. Physiol. 231, 2563–2569. (21) Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T., and Zhao, H. (2015) Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth. Biol. 4, 585–594. (22) Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., and van Ooyen, A. J. J. (2007) High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 73, 4342– 4350. (23) Xie, W., Ye, L., Lv, X., Xu, H., and Yu, H. (2015) Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab. Eng. 28, 8–18. (24) Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H., and Misawa, N. (1994) Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 58, 1112–1114. (25) Oliveira, C., Teixeira, J. A., Lima, N., Da Silva, N. A., and Domingues, L. (2007) Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger β-galactosidase production. J. Biosci. Bioeng. 103, 318–324. (26) Yamada, R., Taniguchi, N., Tanaka, T., Ogino, C., Fukuda, H., and Kondo, A. (2010) Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb. Cell Fact. 9, 32. (27) Kato, H., Matsuda, F., Yamada, R., Nagata, K., Shirai, T., Hasunuma, T., and Kondo, A. (2013) Cocktail δ-integration of xylose assimilation genes for efficient ethanol production from xylose in Saccharomyces cerevisiae. J. Biosci. Bioeng. 116, 333–336. (28) Liu, H., Wei, Z., Dominguez, A., Li, Y., Wang, X., and Qi, L. S. (2015) CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing,

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repression and activation. Bioinformatics 31, 3676–3678. (29) Hoffman, C. S. (2001) Preparation of Yeast DNA, in Current Protocols in Molecular Biology. John Wiley & Sons, Inc. (30) Rozen, S., and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. Clifton NJ 132, 365–386. (31) Teste, M.-A., Duquenne, M., François, J. M., and Parrou, J.-L. (2009) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Mol. Biol. 10, 99.

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Figure 1: Design of “wicket”. “Wicket” contains universal homology arms (uni-HAL and uni-HAR) for exogenous pathway integration and a Cas9 target site for the induction of a DNA double-strand break. Multi-copies of “wickets” were inserted into the yeast genome by CRISPR-Cas9. Exogenous genes with homology arms can enter the genome through pre-assembled integration and cocktail integration after “wicket” opening by Cas9.

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Figure 2: Multiplex targeting of CRISPR/Cas9 in budding yeast by gap repaired sgRNA method. (a). Gap repaired sgRNA method facilitates high triple targeting efficiency in yeast. I. Co-transformation of three sgRNA plasmids. II. Co-transformation of three sgRNA fragments and a linear vector, which shared about 60 bp overlap (gap repaired sgRNA method). (b). Gap repaired sgRNA method enabled multiple insertions of “wickets” with high efficiency. Eight randomly selected colonies were tested for “wicket” insertion by PCR.

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Figure 3: Pre-assembled integration. (a). Percentage of orange colonies. Error bars indicate the standard deviations from three independent biological replicates. (b). Distribution of copy number of the β-carotene genes among eight randomly selected colonies from transformed SHY123. Error bars indicate standard deviations from three repeated analysis. (c). Schematic representation of the “tandem duplication”. (d). Sequence analysis of the tandem duplication fragment.

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Figure 4: Cocktail integration. (a). Percentage of orange colonies in cocktail integration. Error bars indicate the standard deviations from three independent biological replicates. (b). Carotenoid yield of eight colonies from transformed SHY124. Error bars indicate the standard deviations from two biological replicates. (c). Copy number of CrtE, CrtI, and CrtYB in the eight colonies from panel b Error bars indicate standard deviations from three PCR reactions. (d). Tandem duplication of cocktail integration.

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Wicket: a versatile tool for the integration and optimization of

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