Materials and Methods

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

Strains, media and storage

The Saccharomyces cerevisiae strains used in this work belong to the CEN.PK family (Entian and Kötter 2007; Salazar _et al._2017) and are listed in Table 1. The strains were grown at 30°C in 500 mL flasks containing 100 mL chemically defined medium (synthetic medium, SM) (Verduyn _et al._1992) or yeast extract peptone (YP) medium supplemented with 20 g L−1 glucose (YPD) or 6.8 g L−1 maltose (YPM) in an Innova incubator shaker (New Brunswick Scientific, Edison, NJ) set at 200 rpm. SM contains 3 g L−1 KH2PO4, 0.5 g L−1 MgSO4·7H2O, 5 g L−1 (NH4)2SO4, 1 ml L−1 of trace element solution and 1 ml L−1 of vitamin solution as described in (Verduyn _et al._1992). YP medium contains 10 g L−1 yeast extract and 20 g L−1 peptone. When required, SM was supplemented with 150 mg L−1 uracil, 125 mg L−1 histidine, 500 mg L−1 leucine and/or 75 mg L−1 tryptophan (Pronk 2002).

Table 1.

Strains used in this study.

NameRelevant genotypeParental strainOriginCEN.PK113-7DMAT_a _URA3 TRP1 LEU2 HIS3(Entian and Kötter 2007)CEN.PK2-1CMAT_a _ura3-52 trp1-1 his3Δ(Entian and Kötter 2007)CEN.PK122MATa/MATα(Entian and Kötter 2007)EBY.VW4000MAT_a _ura3-52 trp1-1 leu2-3,112 his3Δ hxt13Δ::loxP hxt15Δ::loxP hxt16Δ::loxP hxt14Δ::loxP hxt12Δ::loxP hxt9Δ::loxP hxt11Δ::loxP hxt10Δ::loxP hxt8Δ::loxP hxt4-1-5Δ::loxP hxt2Δ::loxP hxt3-6-7Δ::loxP gal2Δ stl1Δ::loxP agt1Δ::loxP mph2(ydl247w::loxP mph3(yjr160c::loxP_CEN.PK2-1C(Wieczorke _et al._1999)IMX672_MAT_a _ura3-52 trp1-1 leu2-3,112 his3Δ can1Δ::Spcas9-natNT2_CEN.PK2-1C(Mans _et al._2015)IMX1521_MAT_a _ura3-52 trp1-1 leu2-3,112 his3Δ can1Δ::Spcas9-natNT2 gal2Δ hxt4-1-5Δ hxt3-6-7Δ::ars4 hxt8Δ hxt14ΔIMX672This studyIMX1541MAT_a _ura3-52 trp1-1 leu2-3,112 his3Δ can1Δ::Spcas9-natNT2 gal2Δ hxt4-1-5Δ hxt3-6-7Δ::ars4 hxt8Δ hxt14Δ hxt2Δ hxt9Δ hxt10Δ hxt12Δ hxt13Δ hxt15Δ hxt16ΔIMX1521This studyIMX1812MAT_a _ura3-52 trp1-1 leu2-3,112 his3Δ can1Δ::Spcas9-natNT2 gal2Δ hxt4-1-5Δ hxt3-6-7Δ::ars4 hxt8Δ hxt14Δ hxt2Δ hxt9Δ hxt10Δ hxt11Δ hxt12Δ hxt13Δ hxt15Δ hxt16Δ mph2(ydl247wmph3(yjr160cmal11Δ stl1ΔIMX1541This studyOpen in a separate window

To obtain solid media, 2% (w/v) agar was added. Yeast strains and Escherichia coli cultures were stored by adding glycerol to the cultures to a final concentration of 30% (v/v) and stored at –80°C.

Molecular biology techniques

Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich, St. Louis, MI) according to the supplier's instruction. DNA amplification was performed by PCR using Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA) as previously described (Mans et al._2015). Separation of DNA fragments was done in 1% (w/v) agarose gel (Thermo Fisher Scientific) with SERVA DNA Stain Clear G in 1x TAE buffer (Thermo Fischer Scientific). DNA fragments were isolated from gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). DNA concentrations were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Plasmids were constructed _in vitro with NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, Beverly, MA) according to manufacturer's protocol, but with reaction volumes down-scaled by 4-fold. Colony PCR was performed using DreamTaq PCR Master Mix (2x) (Thermo Fisher Scientific). Following the manufacturer's instruction. Yeast transformation was performed with the LiAc/ssDNA method (Gietz and Woods 2002). Yeast genomic DNA was extracted as previously described (Lõoke, Kristjuhan and Kristjuhan 2011) prior to colony PCR.

Plasmid construction

The double sgRNA method for CRISPR/Sp_Cas9-mediated deletions based on pROS plasmid series (Mans _et al._2015, 2018) was used to construct several sgRNA plasmids. The guide RNA sequences were selected to perform the 21 hexose transporter deletions in a minimal number of transformation rounds. Based on alignments of _HXT gene sequences, considering sequences with AT content above 65% and the absence of strong secondary structures, 11 sgRNAs were designed (sgRNA labelled 1 to 11, Fig. 1B) to yield a kit of six sgRNA-carrying plasmids.

Figure 1.

(A) Overview of the chromosomal localization of the deleted hexose transporters. Genes indicated with the same color were removed in the same deletion round. Red, first round; blue, second round; green, third round. (B) Deletion strategy. The scissors indicate the gene targeted by _Sp_Cas9 editing. The circled numbers indicate the sgRNA used to guide _Sp_Cas9 for editing.

The sgRNA plasmids were assembled in vitro from two DNA parts (using NEBuilder HiFi DNA Assembly Master Mix, New England Biolabs). The first part was the plasmid backbone obtained by PCR with Phusion polymerase, using a single primer (6005, Supplemental Material 1) binding at each of the two SNR52 promoters of either pROS10, pROS14 or pROS16 (Table 2; Mans et al._2018). The second part was a 2 μm fragment surrounded by two sgRNA sequences. This fragment was obtained by PCR amplification (DreamTaq polymerase) of the 2 μm of pROS10 using primers containing the specific 20 bp sgRNA recognition (target) sequence and a 50 bp sequence, homologous to the linearized plasmid backbone. Plasmid assembly was followed by chemical transformation to chemically competent _E. coli XL1-blue cells according to the supplier's instructions (Agilent Technologies, Santa Clara, CA) for storage and plasmid propagation, in Lysogeny Broth (LB) medium with 100 mg L−1 ampicillin.

Table 2.

Plasmids used in this study.

NameRelevant characteristicsAddgene #OriginpRS416CEN6/ARS4 ampR URA3(Sikorski and Hieter 1989)pROS102 μm ampR URA3 sgRNA-CAN1.Y sgRNA-ADE2.Y#107924(Mans et al._2015)pROS142 μm ampR _KlLEU2 sgRNA-CAN1.Y sgRNA-ADE2.Y#107928(Mans et al._2015)pROS162 μm ampR _HIS3 sgRNA-CAN1.Y sgRNA-ADE2.Y#107930(Mans et al._2015)pUDR2112 μm ampR _KlLEU2 sgRNA3-HXT8 sgRNA4-HXT14#113870This studypUDR2142 μm ampR URA3 sgRNA5- HXT13-15-16 sgRNA6-HXT2#113871This studypUDR2172 μm ampR HIS3 sgRNA9-MPH2-3 sgRNA10-MAL11#113872This studypUDR2202 μm ampR KlLEU2 sgRNA8-HXT10 sgRNA7-HXT9-11-12#113873This studypUDR2952 μm ampR HIS3 sgRNA2-GAL2 sgRNA1-HXT4-1-5;HXT3-6-7#113874This studypUDR4182 μm ampR URA3 sgRNA11-STL1 sgRNA11-STL1#113875This studyOpen in a separate window

The E. coli colonies were picked and mixed directly in the DreamTaq PCR mix, containing primers binding to the specific 20 bp recognition sequence (primers 11662-11670, 11756) together with two primers binding in the plasmid backbone (primers 4034 and 5941), confirming the presence of one or two target sgRNAs. This PCR will result in four products, since the sgRNA primer can bind on either side of the 2 μM fragment. The orientation of the 2 μm fragment was confirmed by restriction with FastDigest enzymes (Thermo Fisher Scientific) following the supplier's manual. All primers for plasmid construction, diagnostic PCR and sequencing are listed in Supplemental Material 1.

The plasmids pUDR211 and pUDR220 were constructed by in vitro assembly with the backbone of pROS14 (KlLEU2, Addgene plasmid #107928; Mans et al._2018) and insert fragment amplified with primers containing the _HXT8 (primer 9575) and HXT14 (9577) recognition sequences and primers containing the HXT10 (9576) and HXT9, HXT11, HXT12 (9572) recognition sequences, respectively. Similarly, the plasmids pUDR214 and pUDR418 were constructed with the pROS10 backbone (URA3) (Addgene plasmid #107924; Mans et al._2018) and insert fragment amplified with primers containing spacer sgRNA targeting _HXT2 (9574) and HXT13, HXT15, HXT16 (9573) recognition sequences and insert fragment amplified with primers containing the spacer sgRNA targeting STL1 (primer 13616) respectively (Fig. 1). The plasmids pUDR217 and pUDR295 were constructed with the pROS16 backbone (HIS3, Addgene plasmid #107930; Mans et al._2018) and insert fragments amplified with primers containing spacers sgRNA targeting _MPH2, MPH3 (primer 9579) and MAL11 (primer 9585) recognition sequences and insert fragments amplified with primers containing spacers sgRNA targeting GAL2 and HXT1,3-7 respectively. Plasmid confirmation was performed by Sanger sequencing (using primers 2758, 6197, 8556 and 2918) (BaseClear B.V., Leiden, The Netherlands).

Strain construction

The CRISPR/Sp_Cas9 system with double sgRNA-containing plasmids was used to delete 21 genes in the quadruple auxotrophic strain IMX672 in three transformation rounds (Mans _et al._2015). As described above, six sgRNA expression plasmids were constructed, pUDR211, pUDR214, pUDR217, pUDR220, pUDR295 and pUDR418 (Table 2). Strains were transformed with combinations of two plasmids together with double-stranded DNA fragments (repair DNA) designed to repair the double strand break created by _Sp_Cas9. With one exception, repair DNA was a 120 bp oligonucleotide sequence composed of two adjacent 60 bp sequences homologous to sequences located up- and downstream of the DNA break. To prepare the repair DNA fragments, two 120 bp complementary single-stranded oligonucleotides (Supplemental Material 1) were heated for 5 min at 95°C and then cooled to room temperature. To confirm correct annealing, the dsDNA concentration was measured with Qubit fluorometer 2.0 and Qubit dsDNA BR Assay kit (Thermo Fisher Scientific). Deletion of the adjacent _HXT3, HXT6 and HXT7 resulted in the loss of ARS432. To prevent potential replication problems, the repair DNA carried ARS4, amplified by PCR from pRS416 with primers 9525 and 9526. Transformation of S. cerevisiae was performed as previously described (Gietz and Woods 2002).

The first transformation was accomplished with 1 μg of each plasmid pUDR211 and pUDR295 (Table 2), together with 2 μg of each corresponding repair oligonucleotide (Supplemental Material 1). The gene deletions were confirmed by PCR and plasmids were subsequently removed by growing one positive clone in 100 mL YPM medium at 30°C with aeration until the end of the exponential growth on glucose. This procedure was repeated by inoculating 1 μL of the culture to a new 100 mL YPM flask. At the end of the exponential growth phase, the culture was plated on YPM agar to obtain single colonies. Plasmid loss was confirmed by re-streaking single colonies on plates with selective and non-selective media. A single colony, unable to grow on selective medium, was inoculated in liquid YPM and grown overnight in an incubator at 30°C. The strain was stocked as IMX1521 and used for the second round of transformation, in which 2 μg of each plasmid, pUDR214 and pUDR220, and 2 μg of each corresponding repair oligonucleotide were transformed. The genotype was confirmed by PCR and the plasmids were recycled as described above, resulting in strain IMX1541. This strain was transformed with 2 μg of each plasmid pUDR217 and pUDR418, and with 2 μg of each corresponding repair oligonucleotide. The genotype was confirmed by PCR and the plasmids were removed, resulting in strain IMX1812. For the second and third transformation rounds, liquid and solid cultures were supplied with maltose as carbon source.

CHEF electrophoresis

For chromosome separation, contour-clamped homogeneous electric field (CHEF) electrophoresis was used. The CHEF Yeast Genomic DNA Plug Kit (Bio-Rad, Richmond, CA) was used to prepare 1% agarose plugs as recommended by the supplier. The agarose plugs were placed in a 1% megabase agarose gel in 0.5x TBE buffer (Thermo Fisher Scientific), together with the Lambda PFG Ladder (New England Biolabs). The CHEF-DRIII Pulsed Field Electrophoresis system (Bio-Rad) chilled to 14°C was used with a voltage of 5 V/cm, a pulse angle of 120° and pulse time of 60 s during 28 h followed by a pulse time of 90 s for another 16 h. The CHEF gel was stained in 200 mL 0.5x TBE with 3 μg mL−1 ethidium bromide followed by de-staining in 200 mL 0.5× TBE. Images were taken using a UV transilluminator.

Flow cytometric measurement of DNA content

A sample of an exponentially growing aerobic shake-flasks culture was washed with demineralized water, fixed in 70% ethanol and stored at 4°C. Approximately 1 × 107 ethanol-fixed cells were washed with 50 mM Tris-HCl buffer at pH 7.5. The pellet was suspended in the same buffer supplemented with 1 mg mL−1 RNase A and incubated at 37°C for 2 h. Trypsin was added to a final concentration of 3.3 mg mL−1, and the cell suspension was incubated at 37°C for 2 h. Cells were subsequently washed with 50 mM Tris-HCl pH 7.5 buffer, suspended to a final concentration of 2 × 107 cells per mL in 50 mM Tris-HCl pH 7.5 buffer and stored on ice. A total of 2 × 106 cells were mixed in 50 mM Tris-HCl pH 7.5 buffer with or without 1μM Sytox Green Nucleic Acid stain (Sigma), sonicated at 6 μm peak-to-peak amplitude (MSE Soniprep 150, Fisher Scientific, Loughborough, United Kingdom) for 15 s and stored on ice. Analysis was done on a BD-AccuriTM C6 flow cytometer equipped with a 488 nm excitation laser (Becton Dickinson, Franklin lakes, NJ). A minimum of 1000 events were analyzed in FlowJo v10.4.1 (FlowJo LLC, Ashland, OR) to determine the DNA content of the constructed strains. Exponentially growing aerobic shake-flask cultures of the haploid S. cerevisiae strain CEN.PK113-7D and the diploid strain CEN.PK122 were used to identify the fluorescence intensity corresponding to 1 and 2 N peaks, respectively.

Whole genome sequencing

Illumina sequencing

Genomic DNA of IMX1812, IMX672, IMX1541 and CEN.PK2-1C was isolated using the Qiagen 100/G Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. DNA concentration was quantified using a Qubit® Fluorometer 2.0 with Qubit dsDNA BR Assay kit. The genomic DNA of IMX1812 was used to obtain a 300 cycle paired-end library with insert-size of 550 bp and sequenced in-house on a MiSeq sequencer (Illumina, San Diego, CA) using TruSeq DNA PCR-free library preparation. Sequence data are available at NCBI under Bioproject accession number PRJNA478763.

MinION sequencing

For Nanopore sequencing, a 1D sequencing library (SQK-LSK108) was prepared according to the manufacturer's recommendation, shearing DNA with g-TUBE (Covaris Ltd, Brighton), and loaded onto an FLO-MIN106 (R9.4) flow cell, connected to a MinION Mk1B unit (Oxford Nanopore Technology, Oxford, UK). MinKNOW software (version 1.11.5; Oxford Nanopore Technology) was used for quality control of active pores and for sequencing. Raw files generated by MinKNOW were base called, on a local compute server (HP ProLiant DL360 G9, 2x XEON E5-2695v3 14 Cores and 256 GB RAM), using Albacore (version 1.2.5; Oxford Nanopore). Reads, in fastq format, with minimum length of 1000 bp were extracted, yielding 7.15 Gigabase of sequence with an average read length of 7.3 kb. Sequencing data are available at NCBI under Bioproject accession number PRJNA478763.

De novo assembly

De novo assembly was performed using Canu (v1.4, settings: genome size = 12 m) (Koren et al._2017) producing a 12.16 Megabase genome. Paired-end Illumina library was aligned, using BWA (Li and Durbin 2010), to the assembly and the resulting BAM file (Binary Alignment Map file) was processed by Pilon (Walker _et al._2014) for polishing the assembly (for correcting assembly errors), using correction of only SNPs and short indels (–fix bases parameter). Gene annotations were performed using the MAKER2 annotation pipeline (version 2.31.9) (Holt and Yandell 2011) using SNAP (version 2013–11-29) (Korf 2004) and Augustus (version 3.2.3) (Stanke _et al._2006) as _ab initio gene predictors. S288C EST and protein sequences were obtained from SGD (Saccharomyces Genome Database, http://www.yeastgenome.org/) and were aligned using BLASTX (BLAST version 2.2.28+) (Camacho _et al._2009). Translated protein sequences of the final gene model were aligned using BLASTP to S288C protein Swiss-Prot database. Custom made Perl scripts were used to map systematic names to the annotated gene names.

Analysis of copy number variation

Illumina reads from IMX1812 and CEN.PK2-1C were co-assembled to detect copy number variation between the two strains by applying the Magnolya algorithm (Nijkamp _et al._2012).

Growth tests on solid media

Cells from a frozen stock were inoculated in liquid YPM or SM medium supplemented with maltose, uracil, histidine, leucine and tryptophan and grown aerobically at 30°C, 200 rpm. After 8 h, the optical density at 660 nm was measured with a JENWAY 7200 spectrophotometer (Cole-Parmer, Stone, UK). An appropriate volume of cell suspension was spun down (3000 g, 2 min at 4°C) and washed with sterile water, then transferred to a fresh 500 mL shake flask with an initial OD660 of 0.5 and cultivated for 4 hours at 30°C. After measuring OD660 in triplicate, cells were collected by centrifugation (3000 g, 2 min at 4°C), washed with sterile water and resuspended to a concentration of 107 cells mL−1. From this cell suspension, a 10x serial dilution was prepared in water with final concentration of 103 cells mL−1. Then, 10 μL of each concentration were spotted on selective SM agar plates supplemented with uracil, tryptophan, leucine and histidine with 20 g L−1 glucose, 6.8 g L−1 maltose, 20 g L−1 mannose, 20 g L−1 galactose, or 20 g L−1 fructose as carbon source and grown for 2 days at 30°C.

Growth test in shake flask

All liquid cultures with SM were supplemented with uracil, histidine, leucine and tryptophan and grown aerobically in 500 mL shake-flasks at 30°C, 200 rpm. For growth rate determination, cells were inoculated from a frozen stock culture in 100 mL SM medium with 6.8 g L−1 maltose as carbon source. After 8 h of incubation, optical density at 660 nm was measured with a JENWAY 7200 spectrophotometer. Cells were transferred to an initial OD660 of 0.01 and grown overnight in the same medium. The next morning, exponentially growing cultures were transferred to fresh 100 mL SM maltose medium (initial OD660 ∼0.1) and the OD was monitored over time. For growth tests with glucose as carbon source, cells were washed once in water and inoculated to an OD660 of 0.2 in SM with 20 g L−1 glucose. OD660 was monitored until stationary phase was reached. Specific growth rates were calculated from at least six data points evenly distributed during the exponential growth phase.

Samples were taken and spun down (3 min at 13,000 g) for substrate and extracellular metabolite concentration determination by high-performance liquid chromatography (HPLC) analysis. HPLC analysis was performed with an Agilent 1100 HPLC (Agilent Technologies) equipped with an Aminex HPX-87H ion-exchange column (Bio-Rad, Veenendaal, The Netherlands) kept at 60°C, eluted with sulfuric acid (5 mM, 0.6 mL min−1). Detection of glucose, ethanol and glycerol was performed by a refractive-index detector (Agilent G1362A).

Article TitleA toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

Abstract

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popularSaccharomyces cerevisiaestrain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of theLoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in anS. cerevisiaestrain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, ‘genetically unaltered’ hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from mostS. cerevisiaelaboratory strains in only three transformation rounds.


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