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Current GeneticsMicroorganisms and Organelles ISSN 0172-8083 Curr GenetDOI 10.1007/s00294-018-0831-y

Marker-free genetic manipulations in yeastusing CRISPR/CAS9 system

Inga Soreanu, Adi Hendler, DanielleDahan, Daniel Dovrat & Amir Aharoni

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Current Genetics https://doi.org/10.1007/s00294-018-0831-y

ORIGINAL ARTICLE

Marker-free genetic manipulations in yeast using CRISPR/CAS9 system

Inga Soreanu1 · Adi Hendler1 · Danielle Dahan1 · Daniel Dovrat1 · Amir Aharoni1

Received: 11 March 2018 / Revised: 26 March 2018 / Accepted: 27 March 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

AbstractThe budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applica-tions. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.

Keywords S. cerevisiae · CRISPR/CAS9 · Yeast · Marker-free

Introduction

One of the most commonly used model organisms is the budding yeast Saccharomyces cerevisiae. In the past dec-ades, S. cerevisiae was utilized to study a variety of bio-logical processes including stress response, DNA replica-tion and repair, protein post-translational modifications and signaling (Botstein and Fink 2011; Morano et al. 2012; Bell and Labib 2016; Finley et al. 2012). The vast majority of genetic manipulations in yeast including gene knock-out (KO), gene/promoter replacement and gene tagging is based on the use of selectable markers. These cassettes are inte-grated through homologous recombination (HR) to replace a gene of interest for generating gene KO or to enable gene tagging (Wendland 2003; Schneider et al. 1995). To date, the most widely used antibiotic selection marker in S. cer-evisiae is the kanMX marker cassette (Wach et al. 1994;

Güldener et al. 1996). This cassette allowed the generation of the yeast KO library collection where each of the ~ 6000 ORFs was replaced by kanMX cassette (Giaever et al. 2002). The establishment of synthetic genetic array (SGA) (Tong et al. 2001) and the expansion of the pFA and pRS plasmid systems (Goldstein and McCusker 1999; Taxis and Knop 2006) gave rise to two additional antibiotic-selectable mark-ers, the hphMX and natMX cassettes, that are also widely used in yeast genetics (Lai et al. 2016).

Despite their usefulness, the presence of selectable mark-ers in the final strain can lead to misleading phenotype inter-pretation, due to the neighboring gene effect (Ben-Shitrit et al. 2012). In addition, complex genetic manipulations that require the use of multiple selectable markers may be limited by the number of available marker cassettes. Such limitations led to establishment of marker recycling meth-ods including the inducible Cre–loxP recombination system (Güldener et al. 1996), the hisG repeats (Akada et al. 2002) and the URA3-selectable marker (Akada et al. 2006; Wang et al. 2017) that allow excision of the marker gene. However, it has been shown that continuous utilization of these pop-out cassettes can cause decrease in correct integrations and chromosomal rearrangement of the yeast genome (Delneri et al. 2000; Akada et al. 2006).

In recent years, a significant breakthrough in genome editing has been achieved by the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas system (CRISPR/CAS9) (Barrangou et al. 2007). This

Communicated by M. Kupiec.

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0029 4-018-0831-y) contains supplementary material, which is available to authorized users.

* Amir Aharoni aaharoni@bgu.ac.il1 Department of Life Sciences and the National Institute

for Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Be’er Sheva, Israel

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system enables highly accurate and efficient genome editing in a wide variety of model organisms, using a guide RNA (gRNA) that directs the CAS9 endonuclease to perform dou-ble-strand cleavage at a specific genomic locus (Brouns et al. 2008). CAS9 cleavage significantly increases the probability of HR, enabling the site-specific integration of a variety of different donor DNAs with such high efficiency that selec-tion is not needed (Cong et al. 2013; Mali et al. 2013). This system was recently adapted for general use in S. cerevisiae and enables marker-free editing of the yeast genome (Dicarlo et al. 2013; Laughery et al. 2015; Jessop-Fabre et al. 2016) with almost no off-target effect that considered to be unlikely in a small genome as of S. cerevisiae (Ryan et al. 2014; Jakočinas et al. 2015). In the last few years, many studies further developed and applied the CRISPR/CAS9 system for diverse applications in yeast mainly focusing on optimiza-tion/generation of biosynthetic pathways (Ryan et al. 2014; Jakočiunas et al. 2015; Shi et al. 2016, Si et al. 2017) and transcriptional reprogramming (Zalatan et al. 2015; Apel et al. 2017; Elison et al. 2017; Jensen et al. 2017). These applications were performed through multiplexing (e.g., simultaneous genome editing at multiple sites), introduc-tion of large chromosomal segments, multiple genes or point mutations (Bao et al. 2015; Lee et al. 2015; Mans et al. 2015; Walter et al. 2016; Satomura et al. 2017, Si et al. 2017).

One drawback of the CRISPR/CAS9 system is that cleav-age efficiency varies greatly depending on the sequence of the gRNA, and identifying an efficient gRNA for a given tar-get may be laborious. In recent years, numerous studies have attempted to define rule sets that predict gRNA efficiency (Doench et al. 2014, 2016) and create tools for streamlin-ing efficient gRNA design (Haeussler et al. 2016). However, these efforts have mostly focused on gRNA efficiency in

mammalian cells, and their conclusions do not necessar-ily extend to the use of CRISPR/CAS9 in other species (Haeussler et al. 2016). In the absence of equivalent data for yeast, the widespread application of CRISPR/CAS9 in yeast genetics requires a generalized approach which relies on pre-designed gRNAs for the general use of the yeast community.

Here, we combine the power of antibiotic resistance cas-settes with the CRISPR/CAS9 system, to enable fast and efficient genome editing in yeast. We specifically target the CRISPR/CAS9 system for the cleavage of the three com-monly used antibiotic-selectable marker cassettes in yeast (kanMX, natMX and hphMX) for their replacement with a desired donor DNA. The exploitation of highly efficient gRNAs (instead of engineering specific gRNAs for each individual gene) enables the yeast community to utilize previously generated strains as starting points for simple cloning-free genome editing. We demonstrate this approach for marker-free gene/promoter replacements and for increas-ing the efficiency of site-specific integration of a large and repetitive DNA construct (Fig. 1).

Materials and methods

Plasmid generation for CRISPR/CAS9 genome editing

All plasmids were generated based on the backbone of previ-ously published pCAS plasmid [a kind gift from Jamie Cate (Ryan et al. 2014), Addgene plasmid #60847]. The pCAS plasmid was digested by BglII and XhoI to clone the URA3 yeast marker and ampicillin resistance cassette instead of the original kanMX cassette. Both genes were PCR amplified

Fig. 1 CRISPR/CAS9-targeting antibiotic resistance markers (kanMX is shown as an example) in yeast mediates the marker-free genetic integration of donor DNA for different types of genetic manipulation in yeast. Three different applications are shown: gene replacement

(left), promoter replacement (middle) and large array cassette inte-gration (right). These applications are further  described below and shown schematically in more details in Figs. 3, 4 and 5

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and cloned into the plasmid backbone by Gibson Assembly according to the manufacturer’s procedure (NEBuilder). In addition, the gRNA from the original pCAS backbone was replaced by a recognition site for StuI. To add a second gRNA cassette containing HpaI site for downstream clon-ing, the new vector was digested by XhoI and SalI, followed by cloning of the gRNA cassette through Gibson assem-bly. This general plasmid containing two gRNA cassettes was later used for cloning of specific gRNAs targeting the kanMX, natMX and hphMX cassettes. The pCY5k, pCY5n and pCY5h plasmids containing gRNAs specific for kanMX, natMX and hphMX cleavage, respectively, were generated by cloning the different gRNAs to the vector by StuI digestion followed by Gibson assembly. The pCY5.1nk and pCY5.1nh containing two gRNAs targeting the natMX and kanMX and natMX and hphMX, respectively (Table 1), were prepared by digestion of pCY5n with HpaI and cloning of the suitable gRNA through Gibson assembly. The pCY5.1kh plasmid containing two gRNAs targeting the kanMX and hphMX cassettes was prepared by digestion of pCY5k with HpaI and cloning of gRNA targeting the hphMX cassette through Gibson assembly. Strains and plasmids are available upon request. Table 1 contains description of all plasmids gener-ated in this study.

CRISPR/CAS9-mediated gene/promoter replacement

To substitute the YAP1 gene by its orthologues, yap1 dele-tion was generated on the background of BY4741 strain using natMX cassette based on HR followed by selection on YPD containing 100 µg/ml cloNAT. Positive colonies were verified by sequencing of the genomic area. Next, the KO strain was transformed at logarithmic stage with 800 ng of pCY5n and ~ 7 µg of YAP1 PCR product (dif-ferent orthologue each time) and plated on Sc-Ura plates. Colonies that exhibited sensitivity to cloNAT were further verified by sequencing for complete gene replacement. Replacement of the YAP2 gene was performed in the same manner excluding the initial KO. In this case we used a library strain containing yap2 deletion by kanMX cassette (yap2Δ::kanMX) on BY4741 genetic background (Giaever

et al. 2002). The pCY5k was utilized for the Cas9-mediated YAP2 orthologues integration. For promoter replacement, the promoter of CLN2 (ChrXVI:66114–66613, constituting 500 bp prior the ATG of CLN2) was KO by natMX cassette as described above. The KO strain was then transformed at logarithmic stage with the pCY5n plasmid and SVS1 pro-moter PCR product (ChrXVI:241418–241917, constituting 500 bp prior to the ATG of SVS1) containing 30 bp homol-ogy to the appropriate CLN2 genomic region and plated on Sc-Ura plates. Colonies that exhibited sensitivity to cloNAT were further verified by sequencing for complete promoter replacement (Fig. S1). Sequences of all genes used are shown in sequence list, Supplementary Information.

Oxidative stress tolerance assay

Hydrogen peroxide sensitivity analysis of the strains expressing the different YAP1 and YAP2 variants was per-formed using spot assay. The yeast strain cultures were grown overnight at 30 °C, then washed twice and ten times serially diluted from OD600 = 0.6 to OD ~ 0.6 × 10−5. A spot of 5 µl from each of the serially diluted cells was plated on YPD containing 2 mM of H2O2 plates and incubated at 30 °C for 48 h.

Western blot

yap1Δ strain was transformed with a centromeric pRS313 plasmid expressing either the ScYAP1 WT or CaYAP1 ortholog gene fused to a 3xFLAG tag under the native pro-moter. Yeast extracts were generated from 250 ml of loga-rithmic cultures (OD600 of 0.6) using conventional methods. Briefly, cell pellets were lysed with Cell Lytic (Sigma), sup-plemented with protease inhibitors (Sigma) and glass beads, as suggested by the manufacturer. Following centrifuga-tion, cell extracts were collected and protein concentration was determined by the BCA method (Thermo Scientific). Approximately 10 µg/µl of total protein was loaded to 10% SDS-PAGE and analyzed by western blot analysis using primary antibodies against the 3xFLAG tagged ScYAP1 or CaYAP1 (anti-FLAG 1:1000, Sigma) and against pgk1 (1:4000, Invitrogen) that used as loading control, followed by detection using α-mouse HRP-conjugated secondary anti-bodies (1:8000, Jackson).

Alpha-factor synchronization

Single colonies of the strains containing the WT or the strains containing the different promoters were grown over-night in 5 ml YPD at 30 °C, then the cultures were diluted to OD600 ≈ 0.1 in 15 ml YPD and grew at 30 °C to OD600 ≈ 0.4 (logarithmic stage). Next, 100 ng/ml of alpha-factor was added and the cultures were grown at 30 °C for another 2 h

Table 1 List of plasmids generated in this study

Plasmid Target gene

pCY5k (gRNA kanMX) kanMXpCY5h (gRNA hphMX) hphMXpCY5n (gRNA natMX) natMXpCY5.1kh (gRNA kanMX-gRNA hphMX) kanMX and hphMXpCY5.1kn (gRNA kanMX-gRNA natMX) kanMX and natMXpCY5.1nh (gRNA natMX-gRNA hphMX) natMX and hphMX

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(Stuart and Wittenberg 1995). Synchronization was checked by microscope to identify unbudded shmoo-shaped cells. Synchronized cells were washed two times in fresh YPD to delete the alpha-factor and continued to grow in fresh YPD media at 30 °C.

RNA extraction and reverse transcriptase qPCR analysis

Cells were synchronized by alpha-factor arrest and release (see above) and 1.5 ml cells were collected at different time points (0, 30, 60 min) and the pellet was frozen in liquid nitrogen and stored at − 80 °C. Next, total RNA extraction was performed using RNA extraction kit (Quigen) and the total RNA was kept at − 80 °C. Each RNA sample was used as a template for the synthesis of single-strand cDNA by reverse transcriptase (Thermo) using oligo-dT (T18, Sigma Aldrich) as primer. The single-strand cDNA was generated following 1 h incubation at 42 °C and then 5 min at 70 °C. Each 20 µl reaction contained 1500 ng of total RNA and the resulting cDNA was kept at − 20 °C. Relative transcript lev-els were then determined by qPCR analysis using a Bio-Rad PCR. The qPCR was performed in 96-well plates in thermal conditions: 2 min in 50 °C, 10 min in 95 °C, 15 s in 95 °C, and 1 min in 60 °C (those two steps were repeated for 39 cycles). The reaction mix contained 10 µl of SYBR green mix (Applied Biosystem), 10 µM of primers for the quanti-fication of CLN2 (YPL256C), and 20 ng/µl cDNA and DDW to total reaction volume of 20 µl. ACT1 (YFL039C) was used as the internal standard. The primers amplified 101 bp amplicon which was from 405 bp and 333 bp in the ACT1 and CLN2, respectively. The calibration graphs for the CLN1 primers had a slope of − 3.328, the y-intercept is 33.667 and R2 = 0.984; for the ACT1 primers the slope is − 3.425, the y-intercept is 21.785 and R2 = 0.999. The resulting curves represent the averages of at least three independent repeats that were evaluated on the Bio-Rad CFX manager. Table S2 shows the primers used for the qPCR.

pDtet construction and TetO array integration

pDtet was constructed by adding the LEU2 marker and pSR10 target sequences (Rohner et al. 2008) to pUC18 plasmid containing 224 repeats of tetO (Kitamura et al. 2006) using NEBuilder® HiFi DNA Assembly Cloning Kit. The plasmid was digested with EcoRI and KpnI to clone the LEU2 marker (amplified by PCR from pRS315), followed by digestion with SphI and BamHI to clone the pSR10 target sequences (amplified by PCR from pSR10). The pSR10 and pUC18-tetOx224 plasmids were a kind gift from Gasser (Rohner et al. 2008) and Tanaka (Kita-mura et al. 2006) labs, respectively. For array integration, we used the W303 yeast strain expressing a fusion protein

of TetR fused to tdTomato (TetR–tdTomato). Integration of the natMX and URA3 cassette to the target genomic region was performed as in Table 2. The cassette was amplified with both target sequences using primers with homology to the desired genomic location (primers 32–39, Table S2), and extended using primers 40–47 (Table S2) to generate total homology to the genome of ~ 120–140 bp. Array integration without CRISPR/CAS9 plasmid was performed by the trans-formation of 5 µg of AscI-digested pDtet plasmid to expo-nentially growing yeast with an integrated URA3 marker in the chosen location of integration (Table 2). Cells were plated on SC-Leu plates and after 3 days single colonies were transferred to 5-FOA media to validate the loss of the URA3 marker. Clones that grew in 5-FOA-containing media were validated for correct integration of the array by PCR using primers 48–52 (Table S2, Fig. S1). Array integration with the CRISPR/CAS9 plasmid was performed by the co-transformation of 5 µg of AscI-digested pDtet plasmid and 1 µg pCYn (Table 1) to exponentially growing yeast with an integrated natMX marker in the chosen location of integra-tion. Cells were plated on SC-leu-ura plates and after 3 days single colonies were transferred to YPD and YPD + CloNAT plates to validate the loss of the natMX cassette. Colonies that lost the natMX cassette were validated for correct inte-gration of the array by PCR [using primers 48–52 (Table S2, Fig. S1)].

Yeast cell imaging

Single colonies were grown in liquid SC medium and scanned for fluorescent dots in a spinning-disk confocal microscope (Marianas; Intelligent Imaging, Denver, CO) with a 63× oil objective (NA of 1.4) and were imaged using an electron-multiplying charge-coupled device camera (pixel size, 0.079 µm; Evolve; Photometrics, Tucson, AZ).

Results

Design and characterization of the pCY system

To establish a highly efficient system for the generation of new genetically manipulated strains, we have generated a set of six pCY plasmids, based on the pCAS vector devel-oped by Ryan et al. (Ryan et al. 2014), that target CAS9 to cleave the three commonly used antibiotic-selectable mark-ers in S. cerevisiae, the kanMX, natMX and hphMX cassettes (Table 1). In these plasmids both the gRNA and the endonu-clease CAS9 are expressed from a single high-copy 2µ vec-tor. In addition, the plasmids contain the URA3 gene as the selectable marker for quick and easy removal following suc-cessful genome editing by reverse selection on 5-fluoroorotic acid (5FOA) media (see Fig. 2a for a general plasmid map).

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Each plasmid contains a different gRNA targeting one of the three antibiotic cassettes (Fig. 2b), as well as an additional gRNA-expressing cassette containing a HpaI restriction site for the efficient cloning of an additional gRNA for another desired target of interest. In addition, we generated plasmids containing double gRNAs for targeting two marker gene cassettes simultaneously (Table 1). To reduce the probabil-ity for homologous recombination between the two gRNA expression cassettes on the plasmid, these cassettes were cloned on the opposite sides of the selectable marker cas-settes (URA3-Amp).

The efficiency of each gRNA was examined by transform-ing S. cerevisiae strains containing the different cassettes in combination with different linear DNA donors. For exam-ining the plasmids containing gRNA targeting the kanMX, hphMX and natMX resistance cassettes, we utilized PCR fragments of S. cerevisiae YAP2, UBC9 and YAP1 donors (including 50–100 bp homology to the promoter and termi-nator of each gene, Table S2), respectively. The genomic integration was performed in KO strains where the selecta-ble marker cassettes were integrated instead of the native ORF (YAP2, UBC9 or YAP1). We found that 50–100 bp of homology is sufficient for high integration efficiency into the correct genomic locus alleviating the need for complex cloning steps to create long homology regions. Representa-tive positive colonies (e.g., clones that exhibit no growth on selective media) were further sequenced to verify the correct gene integration. All single-gRNA systems ena-bled high-efficiency donor integration and marker cassette replacement with 89, 92 and 99% for kanMX, natMX and hphMX, respectively (Fig. 2c). To verify that gene integra-tion was a result of a CAS9-specific nuclease activity we transformed deletion strains with donor DNA and a pCY

plasmid without a gRNA. As expected, 100% of the colonies exhibit normal growth on selectable media indicating that the donor DNA was not integrated into the genome in the absence of CAS9 cleavage (Fig. S2). Next, we examined the efficiency of a double gRNA guide and focused on the simul-taneous targeting of natMX and kanMX for the integration of YAP1 and YAP2, respectively. The integration was done in the same manner as with a single gRNA in a double-dele-tion strain (yap1Δyap2Δ). The reaction mix included both amplified PCR fragments of ScYAP1 and ScYAP2 (including 50–100 bp homology, Table S2) and a pCY5.1kn plasmid targeting simultaneously the kanMX and natMX cassettes. We found high integration efficiency of 90% of the two YAP genes indicating the feasibility of simultaneous replacement of multiple genes using this system (Fig. 2c and Fig. S3).

CRISPR/CAS9-mediated gene replacement

To demonstrate the applicability of our CRISPR/CAS9-mediated genome editing approach, we first utilized it for YAP1 gene replacement. YAP1 is an AP-1 transcription factor which plays a key role in S. cerevisiae tolerance to oxidative stress (Goldstein and McCusker 1999; Toone and Jones 1999; Morano et al. 2012; Gruhlke et al. 2017). We first generated a yap1 deletion strain (yap1Δ) by substituting YAP1 with the natMX marker cassette. Next, yap1Δ cells were transformed with pCY5n plasmid (Table 1) in com-bination with different donor YAP1 orthologues amplified by PCR (Fig. 3a). This approach allows obtaining marker-free yeast strains containing different YAP1 orthologues through a cloning-free process. As proof of concept we decided to substitute the WT gene by the Candida albicans (CaYAP1) ortholog that originate from clade II of the yeast

Fig. 2 Basic design and characterization of the pCY5 plasmids. a Schematic representation of the pCY5 plasmid based on the pre-viously published pCAS backbone (Ryan et  al. 2014). All plasmids contain a 2µ origin of replication for obtaining a high copy number in yeast, CAS9 gene under the control of a constitutive promoter and URA3 selectable marker. The plasmids include a specific gRNA (gRNA1) for a selectable marker (kanMX, natMX or hphMX) and a restriction site for the cloning of a second gRNA (gRNA2). b Sche-matics of the three antibiotic resistance cassettes where the relative cleavage site is highlighted in white and the specific gRNA sequence

targeting each antibiotic resistance cassette is highlighted above. The Protospacer Adjacent Motif (PAM) is highlighted in red and the estimated cleavage site is marked using a triangle. c Efficiency of CRISPR/CAS9-mediated donor DNA integration (donors are described in detail in the text) using the respective pCY5 plasmid (Table 1) and the gRNA shown in panel  (b). Orange bar shows the efficiency of CRISPR/CAS9-mediated simultaneous integration of two donor DNA integrations. The data presented are the average of three independent transformations while the error bars represent the standard deviation from the average

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phylogenetic tree (Wapinski et al. 2007). Previously, we have utilized this approach to systematically follow the co-evolu-tion of proteins including transcription factors through fungi evolution (Zamir et al. 2012; Sandler et al. 2013; Hendler et al. 2017). The similarity of YAP1 orthologues to ScYAP1 is as low as 50%, while the differences are spread all along the gene. Thus, such ScYAP1 replacement is extremely dif-ficult to perform using the standard CRISPR/CAS9 approach and would require extensive usages of specific gRNAs.

Growth analysis of the different strains under oxidative stress showed that the yap1Δ strain exhibits high sensitivity on H2O2-containing media (Fig. 3b), in agreement with pre-vious studies (Gruhlke et al. 2017). To test whether resist-ance to oxidative stress is increased due to complementation with a YAP1 ortholog, we tested the sensitivity of the strain

expressing CaYAP1 to oxidative stress. We found a decrease in the complementation level of the CaYAP1 ortholog in comparison to ScYAP1. (Fig. 3b). While knock-in of ScYAP1 leads to a full complementation, a strain expressing CaYAP1 shows only a partial complementation. To rule out the pos-sibility that partial complementation of CaYAP1 is due to decrease in protein expression we performed a western blot analysis that confirmed similar protein levels of ScYAP1 and CaYAP1 (Fig. S4).

CRISPR/CAS9-mediated promoter replacement

To examine the applicability of our approach for marker-free promoter replacement, we chose to switch between gene promoters that are controlled by the Swi4 transcription fac-tor. Swi4 is part of the SCB (Swi4 Cell cycle Box) complex and together with the MCB (MluI Cell cycle Box) complex is responsible for the G1-to-S transition in the cell cycle (Bähler 2005). Two known and validated targets of Swi4 are the CLN2 and SVS1 (Ferrezuelo et al. 2010). CLN2 is a G1 cyclin involved in regulation of the cell cycle (Had-wiger et al. 1989) while SVS1 is a cell wall and vacuolar protein important for yeast budding (Nakamura et al. 1995). To examine the effect of promoter location on the periodic expression level of these targets, we utilized the CRISPR/CAS9 system for a marker-free CLN2 to SVS1 promoter replacement. As a first step for this promoter replacement, we deleted the CLN2 promoter region using a natMX cas-sette. Next, cells containing the cln2 promoter deletion were transformed with the pCY5n plasmid together with the PCR-amplified SVS1 promoter containing suitable homology to the CLN2 locus (30 bp homology, Table S2). Such trans-formation allowed the generation of a marker-free strain in which the CLN2 promoter is replaced by SVS1 promoter (Fig. 4a).

To examine the functional consequences of promoter replacement, we performed real-time PCR analysis on RNA extracted from cells containing either the WT CLN2 promoter, cln2-deleted promoter or the SVS1 pro-moter replacing CLN2 promoter. Cells from the different strains were arrested in G1 using alpha-factor followed by a release to rich media and cell samples were collected at different time points (see details in “Material and Meth-ods”). Analysis of the WT strain showed the characteristic periodic expression of CLN2 while the strain containing cln2 promoter deletion showed low CLN2 expression at all time points (Fig. 4b). In contrast to the cln2-promoter-deleted strain, analysis of the strain in which the CLN2 promoter is replaced by SVS1 promoter indicated the rees-tablishment of CLN2 periodic expression similar to the WT strain. Moreover, we found that this strain shows an even higher CLN2 expression 30 min after alpha-factor release indicating the higher efficiency of SVS1 promoter

Fig. 3 CRISPR/CAS9-mediated gene replacement of YAP1. a Sche-matic representation of the CRISPR/CAS9-mediated YAP1 gene replacement with an orthologous gene. The WT strain contains a KO with a selectable marker (top, natMX cassette). Next, cells are transformed with the pCY5n plasmid (Table  1) and a linear donor DNA containing YAP1 orthologue flanked by ~ 100 bp of homology to ScYAP1 promoter and terminator region (middle). Integration of the donor DNA is mediated by the CRISPR/CAS9 cassette cleav-age. Selection on plates lacking uracil allows the isolation of colonies containing a marker-free YAP1 gene replacement (bottom). b Sensi-tivity of the different yeast strains to oxidative stress. Spot assay (see methods for details) of yeast cells plated on YPD plates (left) or YPD plates containing 1.5  mM of H2O2 (right) shows high sensitivity of the yap1Δ strain in comparison to the WT strain (second row). Rein-tegration of the ScYAP1 leads to a full recovery of resistance to oxi-dative stress (third row) while integration of CaYAP1 leads to partial recovery to oxidative stress (row 4)

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to induce CLN2 expression relative to the native promoter (Fig.  4b). Thus, our results show the functionality of the SVS1 promoter following its marker-free integration mediated by the CRISPR/CAS9 system. Further applica-tion of our system provides many possibilities for pro-moter replacements including switching between native

promoters and replacement with orthologues or mutated promoters.

CRISPR/CAS9-mediated operator array integration

Next, we tested the utility of our system for large operator array integration into the yeast genome. Operator arrays are composed of multiple repeats of bacterial operator binding sequences (LacO or TetO) allowing binding of many copies of the bacterial LacI or TetR repressor proteins expressed in yeast. By fusing LacI or TetR to different functional proteins this approach allows their recruitment to a specific location in the yeast genome (Hediger et al. 2004; Löiodice et al. 2014). One popular application of this approach is the fusion of LacI or TetR to fluorescent proteins, a methodology termed fluorescent protein-tagged repressor-operator sys-tem (FROS), this approach is widely used for chromosome tagging, and studying chromatin organization and mobility (Hediger et al. 2004; Löiodice et al. 2014).

A powerful approach for the genome integration of the operator array is by replacing a selective marker located at the desired position with the array fused to a different marker (Rohner et al. 2008). Using this approach, array integra-tion is facilitated by HR of target sequences located at the 5′ and 3′ ends of the linearized plasmid and the genomic marker enabling cloning-free site-specific array integra-tion (Fig. 5a). However, the low integration efficiency at the correct locus is a significant drawback of this approach. We found that the average integration efficiency of an array cassette containing 224 repeats of TetO, determined at two different locations, is only 1.6% (Table 2). Thus, we examined whether the CRISPR/CAS9 system can be uti-lized to significantly increase the site-specific integration of the operator array. Utilizing a yeast strain marked with the

Fig. 4 CRISPR/CAS9-mediated CLN2 promoter replacement. a Schematic representation of the CRISPR/CAS9-mediated CLN2 promoter replacement with the SVS1 promoter. First, the native pro-moter is replaced by antibiotic resistance cassette (top, natMX cas-sette). Next, cells are transformed with the pCY5n plasmid (Table 1) and a donor DNA containing SVS1 promoter flanked by ~ 100 bp of homology region (middle) to the target CLN2 locus. Strain contain-ing a marker-free integration of the SVS1 promoter leading to CLN2 promoter replacement is isolated (bottom). b Periodic expression analysis of CLN2 in WT (red), cln2-promoter-deleted strain (purple) and strain containing SVS1 promoter instead of the native CLN2 pro-moter (green). A characteristic synchronized expression of CLN2 is observed in the WT and the promoters switch strain (red and green), whereas CLN2 expression is very low in the cln2-promoter-deleted strain (purple). The expression data are the average of three inde-pendent repeats while the error bars represent the standard deviation from the average

Table 2 Statistics of TetO array integration at different genomic loca-tions

a A plasmid containing a TetO array was integrated into the genome at various locations, while replacing a natMX cassetteb Fraction of colonies that lost the genomic marker out of the total number of colonies that gained the array marker. This indicates the efficiency of integration at the correct site

Locationa Array plasmid integration efficiencyb

Without CRISPR With CRISPR

chrIV:1185910 1/782/192

16/16

chrVI:221990 0/375/192

24/24

chrIV:344119 ND 15/16chrIV:362562 ND 16/16Average integration

efficiency1.6% 98.6%

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natMX cassette at the target location, we co-transformed the cells with pCY5n plasmid together with a linearized plasmid containing the array and homology regions to the natMX cassette. This transformation allowed the site-specific inte-gration of the array replacing vast majority of the natMX cassette (Fig. 5a). We found that the CRISPR/CAS9 array integration efficiency at four independent locations was 98.6% showing a dramatic increase over the non CRISPR/CAS9 integration efficiency of 1.6% (Table 2; Fig. 5b).

To examine the functionality of the array in yeast, we performed array integration into a yeast strain expressing

TetR fused to tdTomato fluorescent protein (TetR–tdTo-mato). Due to the high level of operator repeats within the array, possible undesired recombination can lead to array shortening and, as a consequence, a decrease of localized red fluorescent signal. As expected, fluorescence microscopy analysis of cells expressing TetR–tdTomato prior to array integration indicated a diffuse fluorescent signal located in the yeast nuclei (Fig. 5c). In contrast, upon array integration, the TetR–tdTomato fluorescent signal becomes highly localized and is clearly observed as a bright red dot marking the yeast genome indicating the

Fig. 5 CRISPR/CAS9-mediated array integration. a Schematic rep-resentation of the site-specific CRISPR/CAS9-mediated Tet operator (TetO) array integration. The array plasmid size is ~ 15 kb containing 224 repeats of TetO sequence and a selectable marker for LEU. First, antibiotic resistance cassette is integrated at a specific chromosomal location (top, natMX cassette). Next, cells are transformed with the pCY5n plasmid (Table 1) and the array plasmid containing homology regions to the natMX cassette to enable array integration (middle). High-efficiency integration of the array at a specific locus (Table 2) allows the fast isolation of the correct strain (bottom). b Comparison of array integration efficiencies in the absence (−pCY5) and in the presence (+ pCY5) of CRISPR/CAS9-mediated array integration. A

dramatic increase of correct site-specific array integration is observed (data are adapted from Table 2 and is the average of three independ-ent repeats while the error bars represent the standard deviation from the average). c Confocal fluorescent and bright-field microscopy images (overlap) of yeast cells expressing TetR fused to tdTomato fluorescent protein (TetR–tdTomato) that do not contain the TetO array. The red fluorescent signal is diffused in the nucleus. d Images of yeast cells containing the TetO array and the TetR–tdTomato fluo-rescent proteins. The array containing 224 TetO sequences leads to a highly localized fluorescent signal that is viewed as a bright fluo-rescent dot allowing a site-specific marking of the DNA for various applications (see text for details)

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maintenance of array integrity following CRISPR/CAS9-mediated integration (Fig. 5d).

Discussion

Marker cassettes are heavily utilized in yeast genetics and thus, thousands of marked yeast strains are available for the usage of the yeast community in addition to strains contain-ing natural variation in their genomes (Cubillos 2016). The simple integration of marker cassettes through HR enables a wide variety of genetic manipulations where the most com-mon ones are gene/promoter KO and tagging of a protein of interest. Here, we combined the power of marker cassette integration with the CRISPR/CAS9 technology to enable common yeast genetic manipulations through a cloning-free process. This combination allows the generation of marker-free strains containing only the gene/promoter replacement without any undesired addition/deletions to the yeast genome (Fig. 1). In addition, we show how this approach can increase the efficiency of integration of large and highly repetitive constructs. Our designed set of plas-mids allows performing these tasks without cloning of the donor DNA and, importantly, does not require the design, cloning and testing of specific guide RNA for the target site in the genome. This approach can be easily expanded for the generation of plasmids containing gRNAs targeting all common marker cassettes used in yeast including the HIS3, TRP1 and LEU2 for various applications. Thus, we believe that this approach will significantly increase the usage of the CRISPR/CAS9 technology enabling the generation of many marker-free strains for a wide variety of yeast genetic studies.

While current CRISPR/CAS9 technologies are highly useful for the generation of point mutations through the generation of specific guides or CRISPR nickase (Satomura et al. 2017), it is extremely difficult to introduce many mutations spread along the gene of interest (e.g., in the case of gene replacement by orthologues). Our approach that can take advantage of existing strains containing anti-biotic resistance cassettes allows the fast replacement of the whole gene/promoter of interest for the targeted integration of a highly mutated gene/promoter of interest. To perform this task, the plasmids reported in this study (Table 1) can be directly utilized with the desired mutated gene/promoter that functions as the donor DNA. As described for other CRISPR/CAS9 applications (Dicarlo et al. 2013; Jakočiunas et al. 2015; Laughery et al. 2015) the efficiency of donor DNA integration is extremely high with no frame shifts or additional mutations in the newly integrated gene/promoter (Fig. 2 and Fig. S1–S2).

Our study complements previous studies that developed approaches for using endonuclease (Khmelinskii et al.

2011) or induced DSB (Storici and Resnick 2006) to pro-mote high level of recombination enabling various yeast genetic manipulations including seamless gene tagging. In addition, a variety of studies utilized the CRISPR/CAS9 technology as a tool for genome editing in yeast. Currently, major applications of this technology in yeast are multi-gene disruption and large deletions (Bao et al. 2015; Hao et al. 2016), introduction of biochemical pathways or other foreign genes into yeast (Ryan et al. 2014; Jakočiunas et al. 2015; Mans et al. 2015) and transcriptional analysis and reprogramming (Zalatan et al. 2015; Apel et al. 2017; Eli-son et al. 2017; Jensen et al. 2017). Approaches for foreign DNA/gene introduction into yeast utilizes different inte-gration sites including delta sites (repetitive sequences that are spread in the yeast genome) (Bao et al. 2015, Si et al. 2017), marker cassettes (Jakočiunas et al. 2015) and other targeted sites (Mans et al. 2015). While extremely pow-erful, these approaches are mainly utilized for synthetic biology applications or for the study of completely foreign genes in yeast (e.g., human or bacterial genes) (Lee et al. 2015; Tsarmpopoulos et al. 2016).

Our approach is mainly suitable for yeast geneticists who are interested to study the function of their favorite gene/promoter through multiple mutations in a marker-free manner. In addition, the application of our approach for increased array integration efficiency is especially important due to the difficulty in obtaining successful strains. During array transformation the array may become shorter by HR due to its repetitive nature and when used for fluorescent marking of the genome, this may result in weak fluorescent signal. Thus, high insertion efficiency is important for the generation of several candidates for the selection of optimal yeast strain with bright dots for further microscopy studies. In addition to using the array for fluorescent DNA marking, the repressor can be fused to any protein of interest, forcing its localization to the chro-matin. This approach has been used for the study of DNA damage and repair (Soutoglou and Misteli 2008; Piccinno et al. 2017) or checkpoint proteins (Bonilla et al. 2008).

Acknowledgements We thank all the members of the Aharoni’s lab for advices and support. This work was supported by the Israeli Sci-ence foundation (ISF) Grant numbers 2297/15 and 1340/17, Binational Science Foundation (BSF) Grant number 2013358 and the European research training network (ITN, Horizon 2020) ES-cat (722610).

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