CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae.

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CasEMBLR: Cas9-facilitated multi-loci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae Tadas Jakociunas, Arun S Rajkumar, Jie Zhang, Dushica Arsovska, Angelica Rodriguez, Christian Bille Jendresen, Mette L Skjoedt, Alex T Nielsen, Irina Borodina, Michael K. Jensen, and Jay D. Keasling ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00007 • Publication Date (Web): 17 Mar 2015 Downloaded from http://pubs.acs.org on March 19, 2015

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ACS Synthetic Biology

CasEMBLR: Cas9-facilitated multi-loci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae

Tadas Jakočiūnas1, Arun S. Rajkumar1, Jie Zhang1, Dushica Arsovska1, Angelica Rodriguez1, Christian Bille Jendresen1, Mette L. Skjødt1, Alex T. Nielsen1, Irina Borodina1, Michael K. Jensen1*, and Jay D. Keasling 1,2,3,4 1

The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark,

Denmark 2 3 4

Joint BioEnergy Institute, Emeryville, CA, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Department of Chemical and Biomolecular Engineering & Department of Bioengineering

University of California, Berkeley, CA, USA

* Corresponding

author: [email protected]

Abstract Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and markerfree genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multi-loci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR, and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

Keywords: DNA assembly, CRISPR/Cas9, double-strand break, metabolic engineering

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Introduction

The yeast Saccharomyces cerevisiae serves as a platform organism for functional genomics of eukaryotes and bio-based production of an ever-increasing list of chemicals and fuels

1–3.

The native homologous recombination (HR) machinery

of S. cerevisiae has also allowed it to be used in diverse plasmid-based and chromosome integrative cloning efforts with appreciable efficiency 4–7. However, it is widely acknowledged that plasmid-based systems can be unstable 8, and for that reason genome integration is the preferred tactic for most current metabolic engineering efforts on multi-step enzymatic pathway construction 9. Since the pioneering studies on integration of linear fragments with kb-sized homologous ends to chromosome integration sites published in the 1980s

10,11,

the last two

decades have witnessed a number of studies which have continued to develop and improve both HR-based in vivo assembly of DNA fragments and methods for genome integration of heterologous DNA 5,12.

Though yeast’s native HR machinery efficiently recombines homologous sequences, the background efficiency for genome integration of linear DNA fragments in yeast without use of a selection marker is too low to be applicable for strain building. This is due to the fact that in vivo assembly supplies DNA fragments with open ends readily accessible for the HR machinery, but chromosomal integration of these fragments requires recombination of DNA fragments into intact genomic DNA, and is therefore far less likely to occur 12. As the original function of HR include repairing double-stranded breaks (DSBs) in the genome, the efficiency of targeting DNA fragments to chromosomes by HR can be dramatically increased when a DSB is introduced into the genome during transformation 12–14. Using the inducible homing endonuclease I-SceI to target a DSB-mediated integration of a 95 bp linear DNA fragment restoring tryptophan prototrophy, recombination efficiencies of 5-20% were achieved 15. This number was approx. 4000-fold higher than recombination efficiencies without DSBinduced integration of DNA fragments as originally described for the delitto perfetto method 12,15. Likewise, in the CATI method (Combined in vivo Assembly


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and Targeted chromosomal Integration), Kuijpers et al. used I-SceI to induce a single DSB for homology-directed and marker-assisted targeting of 10-15 part assemblies with an efficiency of 95%, compared to merely 5% without the induction of I-SceI endonuclease activity 14.

In the past few years, CRISPR/Cas9 has further improved the toolkit for targeted genome engineering 16,17. In the first reported example of genome engineering in yeast using CRISPR/Cas9, DiCarlo et al. showed that homology-directed repair of a Cas9-mediated DSB could be effectively obtained by co-transforming yeast with 90 bp double-stranded oligonucleotides (dsOligos) and plasmid-based expression of a guide RNA (gRNA)18. Due to the ease and versatility of RNAbased guiding of Cas9 endonuclease activity, CRISPR/Cas9 has continued to improve targeted genome engineering in yeast. This includes restoring uracil prototrophy by single-site integration of a three-part DNA assembly in a urastrain, and the marker-free homology-directed knock-out of multiple genes in one step using short (90-120 bp) dsOligos

19–21.

However, in order to leverage

current genome engineering for robust and balanced multi-step metabolic pathway integration in S. cerevisiae chromosomes, there is a continued need to further exploit the potential of both in vivo DNA assembly and CRISPR/Cas9 multiplex and marker-free genome engineering.

Here we combine recent progress within DNA assembly procedures and efficient targeting of DSBs mediated by multiplex CRISPR/Cas9 genome editing in order to develop a one-step method for marker-free editing of S. cerevisiae genomes at multiple loci using in vivo DNA assembly of standard parts. In this study, we apply the genome engineering method for efficient in vivo assembly of a multiple-gene biochemical pathway and construction of a platform strain for aromatic amino acid biosynthesis. Compared to currently available methods, our method is a rapid and versatile approach to edit genomes at multiple loci.



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Results and Discussion

Rationale and design of CasEMBLR In this study we wanted to apply Cas9-facilitated multi-loci integration of DNA parts into S. cerevisiae chromosomes by combining three recently reported techniques: (i) in vivo DNA assembly using linear DNA fragments

15,18,

5,14,

(ii) homology-directed repair of DSBs

and (iii) our recently established method to

efficiently construct multiplex gRNA expression cassettes used for marker-free multi-loci genome engineering

21.

We named the method CasEMBLR: Cas9-

facilitated multi-loci integration of assembled DNA parts into S. cerevisiae chromosomes, as a reference to the two mutually important methods DNA Assembler and Cas9-facilitated integration of dsOligos into S. cerevisiae chromosomes

5,18.

Compared to current procedures, this method should target

multiple DNA part assemblies into a set of defined loci to further allow versatility in terms of strain design with genes or parts sharing sequence similarities between expression cassettes, without compromising strain stability. Taken together, by combining in vivo assembly and targeted integration into multiple loci mediated by Cas9, our method should allow seamless, marker-free and stable genome engineering.

For this purpose we first used CRISPy, our recently developed in silico gRNA selection tool

21,22,

to identify gRNAs specific to loci of interest (Fig. 1), while

minimizing off-targeting of Cas9 activity. Secondly, we applied constitutive plasmid-borne expression of Cas9 for an efficient and easy one-step transformation of a gRNA-expressing plasmid and parts of interest 18,21. Last, for the sake of versatility we included five fragments for each expression cassette: a promoter, a structural gene, a terminator and two 0.5 kb fragments identical to upstream (US) and downstream (DS) sequences of the DSB, respectively (Fig. 1). All fragment ends share 50 bp overlaps with neighboring fragments, which allows for simple integration of 25 bp overhangs into oligonucleotides used for PCR-based parts amplification (Fig. 1).

CasEMBLR for multi-loci integrative pathway building


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As a proof-of-principle we first applied CasEMBLR to reconstitute the biosynthetic pathway for carotenoid production, which gives S. cerevisiae a visible orange phenotype

23–25.

In S. cerevisiae carotenoids can be efficiently

produced from the mevalonate pathway by expressing the carotenogenic genes crtYB (encoding a bifunctional phytoene synthase and lycopene cyclase), crtI (phytoene desaturase) and crtE (heterologous geranylgeranyl pyrophosphate synthase) from the carotenoid-producing yeast Xanthophyllomyces dendrorhous 26

(Fig. 2a).

Before integrating this three-step pathway we first validated the efficiency of Cas9-mediated DSB and integration of in vivo assembled DNA into each individual locus selected for integration. For this purpose we co-transformed yeast expressing Cas9 with DNA for individual five-part in vivo assemblies and an episomal plasmid expressing a gRNA cassette to guide Cas9-mediated DSB at either ADE2, HIS3,, or URA3 genomic sites. Mutant alleles of ade2- and ura3colonies can be easily screened by color appearance of adenine-deficient colonies (pink) or selected by 5-FOA resistance counter-selection 27, respectively. However, for this proof-of-principle study, efficiencies reported from genotyping of correct assembly and integration of the individual expression cassettes were only based on the representative distribution of phenotypes of transformants (orange/pink/white) appearing on plates selected for Cas9 and gRNA expression (Trp+/Leu+) alone. For all integration loci, multiplex PCR of a minimum of 12 colonies per transformation was used to verify both correct assembly and integration, as the expression cassette and flanking sequences are too large (approx. 2.8-4.4 kb) to be efficiently amplified by a single primer pair during colony PCR. Consequently, colonies without integration of the five-part assembly would generate an amplicon with a size of approx. 1.1 kb from amplification of the native locus (Fig. 2b), whereas a correct assembly and integration should yield two amplicons spanning all six junctions of the five-part assembly, including integration. This is exemplified for assembly and integration of the crtI, crtE and crtYB expression cassettes into sites URA3, HIS3, and ADE2, respectively (Fig. 2b). For correct assembly and integration of the crtYB



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expression cassette in ADE2, positive genotyping results matched the pink ade2phenotype (Fig. 2b-c).

Our genotyping showed that RNA-based guiding of Cas9 at individual target sites was indispensable for obtaining correct integration of five-part DNA assemblies, as no positive transformants were obtained from cells transformed with only DNA parts without the gRNA-expressing plasmid when targeting URA3, HIS3 and ADE2 sites (Fig. 2c-d). For assembly of crtYB into ADE2 without expressing gRNA, 0.2% of the colonies were pink (Fig. 2c), but none of these had the correct genotype. Contrastingly, when co-transforming cells with DNA parts and a gRNAexpressing plasmid we observed substantial differences between the engineering efficiencies we obtained for correct assembly onto the individual loci. Based on genotyping, we obtained engineering efficiencies of 96% and 77% for in vivo assembly and integration of crtYB and crtE expression cassettes in ADE2 and URA3, respectively (Fig. 2d), whereas for crtI integration into HIS3, we only obtained 14.6% (Fig. 2d). Following marker-free assembly and integration of five DNA parts (i.e., one expression cassette) at single targeted chromosomal locus, we made a one-step transformation of Cas9-expressing S. cerevisiae with all 15 DNA parts to be assembled and integrated into the three chromosomal loci: URA3, HIS3, and ADE2. As for single site integrations, genotyping of colonies transformed without a multiplex gRNA plasmid for guiding Cas9 DSBs revealed no correct assemblies and integrations of the three carotenoid pathway gene expression cassettes into the targeted integration sites (Fig. 2d). However, from colonies transformed with both DNA parts and the multiplex gRNA plasmid we obtained an average of 4.5% colonies with correctly assembled carotenoid pathway into the ADE2, HIS3 and URA3 loci (Fig. 2d).

The single-site efficiencies obtained for assembly and integration into ADE2 and URA3 are comparable to those obtained from single-fragment integration to other loci using CRISPR/Cas9

18–21.

This could indicate that assembly is not a

limiting factor for high efficiency genome engineering using DNA parts, whereas the low engineering efficiency obtained for HIS3 could be associated with poor Cas9-mediated DSB induction, known to be critical for obtaining high


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engineering efficiencies

14,15.

Further inspection of the 20 nt gRNA sequence

guiding Cas9 to HIS3 site revealed a 5 nt motif 5’-GGCCC-3’ complementary to 5GGGCC-3’ positioned three nucleotides apart. Using the RNAfold web server

28,

such complementarity is likely to cause secondary structure of the gRNA (Supplementary Figure 1), and thereby lowered guiding efficiency or increased off-targeting of Cas9

29.

However, poor assembly of the crtI expression cassette

could neither be ruled out as the reason for the modest efficiency obtained for this single-site engineering attempt. In order to check if suboptimal in vivo assembly of the crtI expression cassette was also affecting the engineering efficiency of crtI into HIS3, we targeted the in vivo assembly of crtI into the RNAguided DSB of URA3. Here we observed an efficiency of 79.2% (Fig. 2e), which is comparable to the efficiency obtained for assembly and integration of the crtE expression cassette into URA3 (Fig. 2d). This indicates that S. cerevisiae’s native capacity for homologous recombination does not have a sequence bias for the different expression cassette assemblies when using the parameters outlined in this study. In order to test if we could increase efficiency of carotenoid pathway construction using CasEMBLR by changing the HIS3 site, we selected a second gRNA target in HIS3 site (hereafter termed HIS3_2) using CRISPy. Transforming the five parts of the crtI expression cassette together with a gRNA expression plasmid guiding Cas9 to the HIS3_2 position we obtained an engineering efficiency of 79.2%, compared to 14.6% when using the former DSB in HIS3 as integration site (Fig. 2d-e). In line with these results, we obtained an approx. 7fold increase of the engineering efficiency when assembling the three-gene carotenoid pathway from 15 parts, using a triple gRNA expression plasmid targeting assemblies into the HIS3_2 site together with the formerly used ADE2 and URA3 sites (30.6% vs 4.5%, respectively)(Fig. 2d-e).

Taken together, our results show that selection of gRNA target sequence is a key parameter for optimal application of CasEMBLR.

CasEMBLR for strain building without obvious phenotypes Having established a proof-of-principle, we next sought to apply CasEMBLR for one-step genome engineering of strains without obvious selectable phenotypes.



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Also, to further advance the applicability of CasEMBLR for one-step genome engineering, we targeted assembly and integration of DNA parts into two chromosomal loci encoding genes of interest for simultaneous targeted knockout. For this purpose, we addressed the need for yeast cell factories with increased aromatic amino acid (AAA) precursors for improved production of a large number of fine and value-added chemicals including alkaloids and flavonoids

30,31.

AAAs are synthesized via the shikimate pathway, starting with

the condensation of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP)(Fig. 3a). This reaction, catalyzed by 3-deoxy-D-arabino-heptulosonate-7phosphate (DAHP) synthase (ARO4), is allosterically inhibited by tyrosine or high concentrations of phenylalanine or tryptophan

32.

Chorismate mutase

(ARO7), another key enzyme for tyrosine and phenylalanine synthesis, is also feedback inhibited by tyrosine

33,34.

Here, we aimed to relieve these feedback

inhibitions on AAA biosynthesis by assembly of the two tyrosine-insensitive versions of ARO4 and ARO7 (hereafter referred to as ARO4* and ARO7*)(Fig. 3a). To minimize the formation of byproduct in the Tyr/Phe branch, we targeted the integration of Tyr-insensitive ARO4* and ARO7* expression cassette assemblies into the PDC5 and ARO10 sites, respectively, encoding two decarboxylases earlier reported to have the highest decarboxylation rates of phenylpyruvate, a phenylethanol precursor and AAA biosynthesis competitor 30,35.

From our one-step in vivo assembly and marker-free integration of ARO4* and ARO7* expression cassettes into PDC5 and ARO10, we obtained an average engineering efficiency of 58% without any phenotypic selection (Fig. 3b-d). As with the assembly of the carotenoid pathway, we found no positive colonies from cells transformed without the double gRNA-expressing plasmid for directing Cas9 to PDC5 and ARO10 (Fig. 3d).

To validate both method and testbed, we analyzed the capacity of our wild-type and engineered strains for the production of p-coumaric acid, a key precursor for flavonoids, by a plasmid-based expression of a bacterial tyrosine ammonia-lyase (Jendresen et al., unpublished results). Unlike its precursor tyrosine, p-coumaric acid is cell permeable and cannot be further metabolized by yeast cells, making it


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a good readout of the flux towards tyrosine. Here, we observed an approximately four-fold increase in the p-coumaric acid titer in cultures of our engineered strain compared to that in cultures of the TAL-expressing reference strain (Fig. 3e).

Benchmarking CasEMBLR In order to allow for an easy comparison between CasEMBLR and other existing genome engineering methods reported in yeast, we highlight key parameters and engineering efficiencies reported for these methods in Table 1.

The over-arching theme for the work presented in this study, was the development of a multiplex and marker-free genome engineering tool by combining CRISPR/Cas9 and in vivo DNA assembly

6,18,21.

Compared to other

pioneering HR-based genome engineering methods like delitto perfetto and DNA Assembler 5,12,15, CRISPR/Cas9 is the only reported method that allows one-step efficient marker-free genome engineering (Table 1). Though the first reported application of CRISPR/Cas9 in yeast only targeted sites with selectable phenotypes

18,

we and others have reported single-fragment integrations into

multiple sites (1 to 5) without the use of markers or selectable phenotypes 19–21. Ideally, our previous successful CRISPR/Cas9-mediated genome engineering of five loci in one-step 21 may allow for construction of larger metabolic pathways in yeast using CasEMBLR. Studies of the capacity for CasEMBLR in terms of numbers of integration sties and parts to be assembled are currently being performed in our laboratory. Still, with the present results, CasEMBLR is to the best of our knowledge the first reported method to combine in vivo DNA assembly and multiplex CRISPR/Cas9 for versatile strain design and building, while maintaining high engineering efficiencies for most of the targeted sites without the use of markers for assembly integration (Table 1; Fig 2d-e and 3d).

Another parameter to benchmark is the use of DNA parts versus yeast integrative plasmids (YIPs). For stable integration of heterologous DNA several procedures make use of YIPs

36,37.

However, the use of YIPs require flanking

fragment ends (0.1-4 kb) to be constructed and cloned together with expression



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cassettes of interest, for each integration site in order to boost marker-assisted integration 36–38. Though CasEMBLR does not reply on YIPs, each integration site still requires a gRNA-expressing plasmid. However, construction of gRNAexpressing plasmids for single-site targeting only require the cyclic amplification of a donor plasmid using a set of primers with the needed 20 bp gRNA target sequences included, whereas multiplex gRNA plasmids require subcloning

21.

Accordingly, this aspect still limits the use of CRISPR/Cas9-mediated highthroughput strain building, and several efforts are currently targeting the delivery and in vivo processing of gRNA in order to simplify further CRISPR/Cas9 18,39.

Also, it should be mentioned that although the edits introduced by

CRISPR/Cas9 and CasEMBLR are marker-free, plasmid-based expression of Cas9 and gRNA(s) requires markers. However, recycling of these are easily accomplished by a single re-streaking of edited strains onto non-selective medium 21.

Finally, data presented in this study show a high degree of site-specificity with respect to successful integration of DNA assemblies at the sites of DNA damage caused by RNA-guided Cas9 (Fig. 2d-e and Fig. 3d). When targeting multi-loci integration, such discrepancies will naturally affect the overall success rate of genome engineering by CasEMBLR, which we also observe here (Fig 2d-e and Fig. 3c-d). We therefore foresee a need to build more high-performing integration sites, like ADE2, HIS3_2, PDC5 and ARO10, for optimal coupling of one-step multi-loci DSBs and integration of assembled DNA parts into such sites. Consequently, our current efforts seek to address this issue by screening and testing for favorable gRNAs targeting native and synthetic ‘landing pads’ in the S. cerevisiae genome with highest possible specificity and genomic sequence divergence to boost targeting of assemblies and limit mis-integration due to sequence homology between donor ends and off-target chromosomal loci. Applying a set of high-performing ‘landing pads’ together with a complementary set of multiplex gRNA expression plasmids will relieve the need for gRNA plasmid cloning and therefore increase throughput.


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Altogether, CasEMBLR is on par with, and in some approaches superior to, stateof-the-art methods for multi-locus integration of DNA assemblies (Table 1). We therefore envision that CasEMBLR can serve as a platform for multi-loci diversity generation

using

libraries

of

parts

to

improve

sequence-to-function

understanding. Furthermore, as evidenced from our successful carotenoid pathway construction and AAA pathway rewiring from DNA parts, CasEMBLR can be a synthetic biology tool to screen for improved traits of yeast-based microbial cell factory when coupled to adequate non-natural selection or directed evolution regimes. With simple DNA parts amplification/synthesis and easy cloning of multiplex gRNA plasmids, CasEMBLR has impacted the strain engineering procedures in our laboratory, and we believe that CasEMBLR complements current strain building procedures to further develop S. cerevisiae as a preferred cell factory chassis for complex multi-step metabolic engineering strategies.

Methods

Strains, plasmids, media and primers The yeast strains used in this study were isogenic to CEN.PK111-27B. Strains and plasmids are listed in Supplementary Tables 1 and 2, respectively. Strains were grown in complete medium (YPD) with 2% glucose and synthetic complete medium (SC, Sigma-Aldrich), supplemented with 2% glucose, minus the auxotrophic components complemented by propagated plasmids. Cell were propagated at 30°C. All primers used in this study are listed in Supplementary Table 3.

DNA preparation For five-part in vivo assembly, yeast promoters (TDH3p, PGK1p, TEF1p), terminators (ADH1t, CYC1t, VPS13t, PRM9t) and homology sequences were amplified from genomic DNA isolated from S. cerevisiae CEN.PK113-7D using Phusion High-Fidelity Polymerase (F-531L; Thermo Fisher Scientific) following the manufacturer’s recommendations. The structural genes crtE, crtYB, and crtI were amplified from plasmid YIplac211-crtYB/crtI/crtE


26

kindly provided by


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Prof. Gerhard Sandman. The ARO4*(K229L) and ARO7*(G141S) mutants were constructed by site-directed mutagenesis according to sequence requirement for Tyr-insensitivity earlier reported

30,34.

All PCR reactions added the relevant 50

bp overlaps to the ends of each part to allow in vivo assembly and integration by homologous recombination 5. The primers used for amplification are listed in Supplementary Table 3. Following confirmation of their size by agarose gel electrophoresis, the products were individually purified using Nucleospin PCR clean-up columns (740588.250; Macherey-Nagel).

To perform one-step in vivo assembly and integration targeting the locus or loci of interest, 4 picomoles of each part were mixed together, concentrated by ethanol precipitation

40,

and resuspended in 5 μL Milli-Q water. The mixes of

resuspended DNA parts were used with the appropriate gRNA plasmid for electroporation-mediated transformation as described below.

gRNA selection and plasmid construction To select for specific gRNAs targeting ADE2, URA3, HIS3, HIS3_2, ARO10 and PDC5 sites all potential gRNA targets in these annotated CEN.PK113-7D genes were compared to all potential off-targets in the entire CEN.PK113-7D genome using the CRISPy tool (http://staff.biosustain.dtu.dk/laeb/crispy_cenpk/)

21,22.

From this ranking, only gRNAs without any 100%-identity to other genomic loci were selected. gRNA sequences are listed in Supplementary Table 4. To search for predicted secondary structures in the gRNAs selected from CRISPy, we used RNAfold

from

the

Vienna

RNA

suite

(http://rna.tbi.univie.ac.at/cgi-

bin/RNAfold.cgi) 28.

To create a gRNA expression plasmid, first, a backbone vector was constructed. pESC-LEU was amplified with TJOS-97F and TJOS-97R and re-ligated to insert a site compatible for USER cloning and create a cloning vector pTAJAK-96. pTAJAK-96 was digested with AsiSI and Nb.BsmI to create compatible ends for USER cloning. Second, gRNA expression cassettes for all targeted loci were ordered from Integrated DNA Technologies as gBlocks. Sequence of gRNA expression cassette was used as in DiCarlo et al. (2013). Third, gRNA cassettes


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were amplified with universal primers (TJOS-62, TJOS-63, TJOS-64, TJOS-65, TJOS-66, TJOS-67) to attach compatible ends for USER cloning 21. Finally, desired combinations of single (ADE2; URA3; HIS3), double (PDC5-ARO10) and triple (ADE2-URA3-HIS3) gRNA expression cassettes were constructed by USER cloning into an AsiSI-, Nb.BsmI-digested pTAJAK-96 vector 21.

Strain construction The strain expressing Cas9 was constructed by transforming the Cas9 expression plasmid p414 (Addgene reference number: 43802) into CEN.PK111-27B by the lithium acetate transformation method 41. This strain was named TC-50 and used for all further manipulations. To construct a platform strain (PDC5::ARO4*ARO10::ARO7*), TC-50 was transformed with individual five-part mixes (ARO4*, ARO7*; 4 picomoles of each individual part) for in vivo assembly and 1 μg of gRNA plasmid for targeting PDC5 and ARO10 by electroporation 42. The platform strain (PDC5::ARO4*-ARO10::ARO7*) was named TC-49. To test in vivo assembly and integration of carotene biosynthesis genes, strain TC-50 was used for electroporation-mediated transformation with appropriate individual five-part mixes (4 picomoles of each individual part) and with 0.5 μg (for single gRNA), and 1.5 μg (for triple gRNA) of gRNA plasmid for targeting appropriate sites. For no gRNA control, plasmid pTAJAK-96 was used. After transformation cells were plated on SC-TRP-LEU to select for cells carrying Cas9 and gRNA plasmids. To evaluate the tyrosine production, CEN.PK113D-7D reference strain and PDC5::ARO4*-ARO10::ARO7* strains were transformed with a CEN/ARS plasmid pRS415 expressing a tyrosine ammonia-lyase (Jendresen et al., unpublished results) controlled by yeast TEF1 promoter (420 bp) and ADH1 terminator.

Strain genotyping For genotyping, a minimum of 12 colonies from each transformation were analysed. As each expression cassette was too big to amplify using a single primer pair, we performed multiplex colony PCR with two primer pairs for each targeted locus. The multiplex PCR was used to verify (i) if transformants had the correctly assembled cassettes, and (ii) if it was integrated in the loci of interest (Fig. 2b and 3b). The first pair’s forward primer primes 50-100 bp upstream of



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the upstream homology sequence and its reverse primer primes 50-100 bp downstream Start codon of the gene of interest, spanning the homology sequence and promoter in the process. The second pair’s forward primer primes 30-50 bp upstream of the gene’s Stop codon and the reverse primer primes 50100 bp downstream of the downstream homology sequence, spanning the terminator and homology sequence. A correctly assembled gene expression cassette integrated at the correct locus would yield two bands, one 1.4-1.6 kb and the other 700-800 bp depending on the sizes of the terminator and promoter used. If a gene is not assembled at the right locus, the innermost and outermost primers of the mix will amplify approx. 550 bp upstream and downstream of the PAM site in the locus, yielding a single PCR product approx. 1.1 kb on average. Cells from each colony were lysed in Milli-Q water using the protocol and lysis volume recommended for Lyse and Go reagent (Thermo Fisher Scientific)(see also Supplementary Table 5)

43.

Following lysis, the lysate was subjected to 40

cycles of colony PCR using the OneTaq Quick-Load 2xMaster mix with standard buffer (New England Biolabs). The primer concentration for the downstream colony PCR, yielding the shorter product and in general the more efficient of the two PCR reactions was half of that of the upstream colony PCR (200 nM).

HPLC Yeast strains were cultured in 0.5 mL of Feed-In-Time fed-batch medium (M2P Labs) in 96-well plate and shaken at 200 rpm, 30°C for 72 hours. Cells were centrifuged at 2000 g for 15 min, and 150 µl of supernatant was mixed with equal volume of absolute ethanol. p-coumaric acid standards (0.1-1 mM) (SigmaAldrich) were prepared in ethanol (50%, v/v). Quantification of p-coumaric acid was performed on HPLC (Thermo) equipped with a Discovery HS F5 150 mm X 2.1 mm column (particle size 3µm). Samples were analyzed using a gradient method with two solvents: 10 mM ammonium formate pH 3.0 (A) and acetonitrile (B) at 1.5 mL min-1. The program started with 5% of solvent B (0 0.5 min), after which its fraction was increased linearly from 5% to 60% (0.5 – 7.0 min) and held for 2.5 min (7.0 - 9.5 min). Then the fraction of solvent B was decreased back to 5% (9.5 - 9.6 min) and remained until the end (9.6 - 12 min).


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p-coumaric acid was detected by absorbance at 277 nm and the peak (retention time 4.7 min) area was integrated and used for quantification by fitting with a standard curve. All strains were measured in 8 biological replicates (from 8 colonies) and technical duplicates.

Acknowledgement This work was supported by the Novo Nordisk Foundation. Authors would like to acknowledge Prof. Gerhard Sandmann for plasmid sharing. The authors declare that they have no conflicting interests.

Supporting Information: Supplementary Table 1. Strain table Supplementary Table 2. Plasmid table Supplementary Table 3. List of primers Supplementary Table 4. List of gRNAs Supplementary Table 5. Thermo cycler program for yeast cell lysis Supplementary Figure 1. Secondary structure prediction and minimum free energy (MFE) for gRNA targeting HIS3 and HIS3_2 sites

This material is available free of charge via the Internet at http://pubs.acs.org. Figure Legends

Fig. 1. Outline of the CasEMBLR method. To identify gRNAs without predicted off-targets we used the publicly available online tool CRISPy 21,22. Selected gRNAs were cloned into single, double and triple gRNA expressing cassettes for plasmid-based expression. Plasmids expressing gRNA(s) and linear DNA parts with 50 bp overlap were co-transformed into Cas9-expressing S. cerevisiae cells for Cas9-facilitated multi-loci genomic integration of in vivo assembled DNA parts.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 2. Assembly of the carotenoid pathway by CasEMBLR. (a) The mevalonate pathway in yeast and the carotenoid synthesis pathway of X. dendrorhous (orange). (b) Schematic illustration of the five-part assembly of the crtE, crtI, and crtYB expression cassettes and homologous recombination with chromosomal target sites URA3, HIS3, and ADE2. Genotyping primers (arrows) for multiplex PCR are so designed that a correctly assembled and integrated product will yield two PCR products (approx. 1.5 and 0.75 kb), and no assembly yields a single band (approx. 1.1 kb) from the multiplex primers complementary to the flanking sequences (light grey). (c) (Top) Representative phenotype of Cas9-expressing S. cerevisiae transformed with DNA parts for in vivo assembly of the crtI expression cassette and an empty control plasmid (left) or a gRNAexpressing plasmid for DSB-mediated integration of the five-part crtI expression cassette into ADE2 (right). (Bottom) Genotyping result using a nested PCR to identify correct crtI expression cassette assembly and integration into ADE2. On the left, genotyping result of 6 representative colonies from Cas9-expressing cells co-transformed with DNA parts and the empty control plasmid. On the right, genotyping result of 6 representative colonies from cells co-transformed with DNA parts and a gRNA expressing plasmid targeting Cas9-mediated DSB to ADE2. Positive colonies should have two distinct bands the size of approx. 1.5 kb and 0.75 kb, whereas no integration in ADE2 should give a band of approx. 1.1 kb. Full details of the genotyping scheme are provided in the Methods. (d) Average engineering efficiencies obtained from two biological replicate transformations. Error bars represent one standard deviation of the mean. Dashed line separates bars representing single and triple integration efficiencies. (e) Optimization of CasEMBLR by use of a HIS3_2 gRNA. Bars represent average engineering efficiencies obtained from two biological replicate. Error bars represent one standard deviation of the mean. Dashed line separates bars representing single and triple integration efficiencies.

Fig. 3. Construction of an improved tyrosine production strain by CasEMBLR. (a) Schematic representation of S. cerevisiae aromatic amino acid pathway highlighting the genes targeted for the improved tyrosine production strain. (b) Schematic illustration of the five-part assembly of the ARO4* and


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ARO7* expression cassettes and homologous recombination with genomic PDC5 and ARO10, respectively. Design of genotyping primers (arrows) for multiplex PCR are similar to those represented in Fig 2b. (c) Genotyping result using a nested PCR to identify correct assemblies and integration into ARO10 (top) and PDC5 (bottom). On the left, genotyping result of 6 representative colonies from Cas9-expressing cells co-transformed with DNA parts and the empty control plasmid. On the right, genotyping result of 6 representative colonies from cells co-transformed with DNA parts and the double gRNA expressing plasmid targeting Cas9-mediated DSB to ARO10 and PDC5. Positive colonies should have two distinct bands the size of approx. 1.5 kb and 0.7 kb, whereas no integration in ARO10 or PDC5 should give a band of 1.1 kb. Full details of the genotyping scheme are provided in the Methods. (d) Average engineering efficiencies obtained from two biological replicate transformations. Error bars represent one standard deviation of the mean. (e) Average p-coumaric acid titers in CEN.PK reference and PDC5::ARO4*/ARO10::ARO7* strains expressing a bacterial TAL (Jendresen et al., unpublished results) for one-step conversion of tyrosine to pcoumaric acid. Error bars represent one standard deviation of the mean (n=8).

Table 1. Benchmarking CasEMBLR. Comparison of different genome engineering methods in S. cerevisiae.

Supplementary Figure 1. The optimal secondary structure with minimum free energy (MFE) for gRNAs targeting HIS3 and HIS3_2 sites as predicted by the RNAfold web server

28.

The secondary structure is colored by base-pairing

probabilities according to the color key (right). For unpaired regions the color denotes the probability of being unpaired.

References



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(34) Brown, J. F., and Dawes, I. W. (1990) Regulation of chorismate mutase in Saccharomyces cerevisiae. Mol. Gen. Genet. 220, 283–8. (35) Romagnoli, G., Luttik, M. A. H., Kötter, P., Pronk, J. T., and Daran, J.-M. (2012) Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 78, 7538– 48. (36) Siddiqui, M. S., Choksi, A., and Smolke, C. D. (2014) A system for multi-locus chromosomal integration and transformation-free selection marker rescue. FEMS Yeast Res., 14(8): 1171-85. (37) Jensen, N. B., Strucko, T., Kildegaard, K. R., David, F., Maury, J., Mortensen, U. 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, 238–48. (38) Wach, A., Brachat, A., Pöhlmann, R., and Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808. (39) Gao, Y., and Zhao, Y. (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–9. (40) Sambrook, J., and Russell, D. W. (2006) Standard ethanol precipitation of DNA in microcentrifuge tubes. CSH Protoc. 2006. (41) Gietz, R. D., and Schiestl, R. H. (2007) Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 38– 41. (42) Wu, S., and Letchworth, G. J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152–4. (43) Lyse and Go PCR Reagent, Product #78882; Pierce Biotechnology/Thermo Fisher Scientific: Rockford, IL, 2010.


gRNA2

A1 gRN

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gRNA 3


Chr. X Chr. Y Chr. Z

Cas9

Y X

Z gRNA


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Page 23 of 26

Design of gRNAs gRN

CRISPy

A1 gRNA 2

gRN A1

gRNA 2

Chassis

A1

gRNA

3

gRN

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DNA parts

Chr. X Chr. Y Chr. Z Cas9

Down

3

Down

2

Prom 3 Prom

1

Gene 3

Down 1 Gene 2

Ter m

2

Term 3

Prom

2

Up 1

Up 3 Up 2

Gene 1

Term 1

Engineered marker-free chassis

Chr.YY Chr.

Chr. Z

Chr. X Chr. Y Chr. Z

Chr. X



a

c

Acetyl-CoA

- gRNA

+ gRNA

Mevalonate

IPP

DMAPP GPP 1.5 kb 1 kb 0.75 kb

crtYB β-carotene

40 20 0

crtE

URA3_DS

-g rtI

_c

U R A3

H IS 3

rtY

50 bp overlap

_c

CYC1

50 bp overlap

ADH1

50 bp overlap

50 bp overlap

HIS3_DS

HIS3

60 40 20 0

+

_c

ew

ADE2_DS

ca.1100 bp


IS H

B,

_c

E2

_c

ADE2

rtY

rtY

B,

H

IS

3n

50 bp overlap

crtYB

H IS 3

CYC1

AD

ADE2_US

50 bp overlap

U R A3

TEF1

U R A3

_I

-g

ca.1100 bp

80

_I

crtI

100

E2

HIS3_US

TDH3

R N A

e

ca.1100 bp

Engineering efficiency (%)

URA3

AD

URA3_US

PGK1

B

-g

R N A

b

60

-g R N w A rtI _ I+ ,U 3n ew R g R A _c N 3_ A rtI cr ,U tE R gR A3 _c N A rtE + gR N A

Lycopene

crtI

80

_I

Neorosporene

100

w

crtI

ne

d

H IS 3

Phytoene

R N AD _cr A tE E2 -g _ cr AD R N tY E2 A B H _c + IS gR rtY 3_ AD N B, cr A E2 U tI H R _c + IS A3 gR 3_ rtY _ c N c B, rtI rtE A ,U H IS + R gR 3_ A3 cr N _c tI, A rtE U R -g A3 R _c N A rtE + gR N A

Ergosterol

crtYB

gR N A

GGPP

ne

crtE

Squalene

Engineering efficiency (%)

FPP

AD E2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

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a

E4P DAHP

Chorismate Prephenate Phenylpyruvate

b

PEP ARO4

PDC5_US

50 bp overlap

PDC5_DS

PDC5

TDH3

p-Hydroxy-phenylpuruvate

L-Phe L-Tyr

ARO4*

ca.1100 bp

PDC5

Phenylacetaldehyde

ADH1

50 bp overlap

ARO7

PDC5 ARO10

TEF1

ARO10_US

50 bp overlap

VPS13 50 bp overlap

ARO7*

ARO10_DS

ARO10 ARO10

p-Hydroxy-acetaldehyde

ca.1100 bp


5

ce

::A R O 4*

0

c5 /p d 0: :A R O 7*

0

10

en

20

15

o1

1.5 kb 1 kb 0.75 kb

o1

PDC5

40

20

fe r

60

25

ar

80

re

100

p-coumaric acid (μM/OD)

1.5 kb 1 kb 0.75 kb

+ gRNA

e

C EN .P K

ARO10

- gRNA

d Engineering efficiency (%) 0: :A R O - g 7*/ R pdc N A 5:: AR ar O o1 4* 0: :A R O + 7*/ gR pd N c5 A :: AR O 4*

c

ar

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACS Synthetic Biology Method! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Chromosomal integration (no./ transformation)!

Page 26 of 26

No. parts/ site!

Endonuclease!

Selection!

Efficiency!

Ref.!

Single-locus integration, assembly option! Native HR!

1!

1!

-!

+!

10-6 – 10-4!

Wach et al., 1994!

Delitto perfetto, I!

1!

1!

-!

+!

10-5!

Storici et al., 2001!

Delitto perfetto, II!

1!

1!

I-SceI!

+!

20%!

Storici et al, 2003!

CATI!

1!

10-15!

I-SceI!

+!

95%!

Kuijpers et al., 2013!

CRISPRm!

1!

3!

Cas9!

+!

85% (diploid yeast)!

Ryan et al., 2014!

Multi-loci integration, no assembly! EasyClone!

1-3!

1!

-!

+!

44% (3x)!

Jensen et al., 2014!

D-POP!

1-3!

1!

-!

+!

33%!

Siddiqui et al., 2014!

HI-CRISPR!

1 and 3!

1!

Cas9!

-!

100%!

Bao et al., 2014!

Single and Multiple knock-out!

1-5!

1!

Cas9!

-!

100%!

DiCarlo et al., 2013; Jakočiūnas et al., 2015!

-!

+!

20%!

Shao & Zhao, 2009!

30,6% (3x)! 58% (2x)! 97% (1x)!

Jakočiūnas et al., this study!

Multi-loci integration, assembly! DNA Assembler!

>10 (δ-sites)!

4-9!

CasEMBLR!

1-3!

5!

Cas9! -! ACS Paragon Plus Environment

Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

DNA of Saccharomyces cerevisiae.

Ribosomal DNA magnification in Saccharomyces cerevisiae.

A DNA replication enhancer in Saccharomyces cerevisiae.

RNA-DNA sequence differences in Saccharomyces cerevisiae.

Seven-base-pair inverted repeats in DNA form stable hairpins in vivo in Saccharomyces cerevisiae.

In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae.

Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo.

Mutants of Saccharomyces cerevisiae that incorporate deoxythymidine 5'-monophosphate into DNA in vivo.

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae.

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.

Instability of simple sequence DNA in Saccharomyces cerevisiae.

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae.

Characterization of 2-mum DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid.

Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro.

The regulation of mitochondrial DNA levels in Saccharomyces cerevisiae.

YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae.

Error-free DNA-damage tolerance in Saccharomyces cerevisiae.

δ Sequences mediate DNA rearrangements in Saccharomyces cerevisiae.

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae.

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae.

Determination of the molecular weight of Saccharomyces cerevisiae nuclear DNA.