pubmed.ncbi.nlm.nih.gov

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae - PubMed

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Robert Mans et al. FEMS Yeast Res. 2015 Mar.

Abstract

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.

Keywords: CRISPR/Cas9; S. cerevisiae, gRNA; genetic modification; plasmid; webtool.

© Federation of European Microbiological Society 2015.

PubMed Disclaimer

Figures

Graphical Abstract Figure
Graphical Abstract Figure

CRISPR/Cas9 like a Swiss army knife enables molecular biologists to quickly introduce simultaneous multiple and diverse genetic modifications in baker's yeast Saccharomyces cerevisiae.

Figure 1.
Figure 1.

Workflow for CRISPR/Cas9 modification of S. cerevisiae genome using single and double gRNA plasmid series (pMEL10–pMEL17 and pROS10–pROS17, respectively). (a) All oligonucleotides, required for targeting the gene(s) of interest (GOI), can be automatically designed with the Yeastriction webtool (

http://yeastriction.tnw.tudelft.nl

). For the single gRNA method, the tool designs complementary oligonucleotides that can be annealed to form (i) a double-stranded repair fragment and (ii) a double-stranded insert which contains the target sequence for the GOI. For expression of the gRNA, a plasmid backbone containing the genetic marker of choice is amplified from a single gRNA plasmid (pMEL10–pMEL17). Gene deletion is achieved via co-transformation of the plasmid backbone, the dsDNA insert (containing the gRNA target sequence, flanked by sequences identical to both sides of the linearized plasmid backbone) and the repair fragment. For the double gRNA method, Yeastriction designs two sets of oligonucleotides, the first oligonucleotide binds to the 2 μm fragment and has a tail containing the desired target sequence for the GOI and a sequence identical to both sides of a linearized double gRNA plasmid backbone (pROS10–pROS17), the second set of oligonucleotides can be annealed to form the dsDNA repair fragment(s). To construct a gRNA plasmid with two target sequences, first a double gRNA plasmid backbone with the appropriate marker (pROS10–pROS17) is amplified by PCR, excluding the 2 μm fragment. Then, the 2 μm fragment is PCR amplified using the primers harbouring the targets for the GOIs and sequences identical to the plasmid backbone. The final plasmid is then constructed in vitro using the plasmid backbone and the 2 μm fragment, e.g. with the Gibson assembly method. After confirmation of correct plasmid assembly using restriction analysis or PCR, the resulting plasmid is transformed to yeast, together with the appropriate 120 bp dsDNA repair fragment(s). After transformation, the desired genetic modification(s) are checked by PCR, Southern blot analysis or sequencing. Subsequently, the strain can be modified again in a new round of transformation (preferably using plasmids with other markers). Before physiological analysis, the gRNA plasmid(s) are preferably removed. This can be done by growing the strains in liquid media without selection pressure or, if possible, by counter-selection pressure (with 5-fluoroorotic acid, fluoroacetamide or 5-fluoroanthranilic acid for pMEL10+pROS10 plasmids, pMEL11+pROS11 plasmids and pMEL17+pROS17 plasmids, respectively). After confirming plasmid removal by restreaking the same colony on selective and non-selective medium and/or PCR analysis, the resulting strain is re-grown in liquid medium and stored at –80 °C. (b) Architecture of the single gRNA plasmid series (pMEL10–pMEL17). The primers used for PCR amplification of the plasmid backbone are indicated by black arrows. (c) Architecture of the double gRNA plasmid series (pROS10–pROS17) with two gRNA cassettes. The plasmid backbone can be PCR amplified with a single primer (indicated with a black arrow). The 2 μm fragment is amplified with primers designed using Yeastriction (indicated in orange and light green coloured arrows).

Figure 2.
Figure 2.

Efficiency of gene deletion obtained after transformation with a single gRNA plasmid. (a) Quantification of the number of colonies and corresponding gene deletion efficiencies, obtained after transformation of S. cerevisiae IMX672 (ura3–52 trp1–289 leu2–3,112 his3Δ, can1Δ::cas9) with 100 ng pMEL10 backbone, 300 ng of gRNA insert DNA and 2 μg of the corresponding 120 bp dsDNA repair fragment. The transformation targeting MCH1, MCH2 and MCH5 simultaneously was performed using 300 ng of each insert and 2 μg of each repair fragment. For the transformations with repair fragments, the exact number of transformants could not be determined, but exceeded 5000 colonies per plate. The data represent average and standard deviation of transformants of three independent transformation experiments. The estimated total number of colonies carrying gene deletions was based on colony PCR results of 24 randomly picked colonies. In red: percentage of colonies without gene deletions, in blue: percentage of colonies containing one or two but not all deletions, green: percentage of colonies containing all desired gene deletions. No transformants with all three genes deleted were identified. (b) Diagnostic primers were designed outside of the target ORFs to differentiate between successful and non-successful colonies via PCR. In this colony PCR example, primers 6862 & 6863 were used to amplify the MCH1 locus. (c) Example of a diagnostic gel from the transformation targeting the MCH1 locus. The first lane (L) contains the GeneRuler DNA Ladder Mix. Lane 1–8 show the PCR results of eight randomly picked colonies. Successful deletion of MCH1 results in a PCR fragment with a length of 729 bp (lane 1, 3 and 7), when MCH1 is still present a band is observed at 2190 bp (lane 2, 4, 5, 6 and 8).

Figure 3.
Figure 3.

Efficiency of gene deletion obtained after transformation with double gRNA plasmids. (a) Quantification of the number of colonies and corresponding gene deletion efficiencies after transformation of S. cerevisiae IMX672 (ura3–52 trp1–289 leu2–3,112 his3Δ, can1Δ::cas9) with 2 μg of various double gRNA plasmids with 1 μg of the appropriate repair fragment(s). When multiple plasmids were transformed simultaneously, 2 μg of each plasmid was added. The data represent average and standard deviation of transformants of three independent transformation experiments. In red: percentage of colonies containing no gene deletions, in blue: percentage of colonies containing some but not all targeted gene deletions (1, 1–3 and 1–5 respectively), in green: percentage of colonies containing all targeted simultaneous gene deletions (2, 4 and 6 respectively). (b) Diagnostic primers were designed outside of the target ORFs to differentiate between successful and non-successful colonies via PCR. In this colony PCR example, primers 6862 & 6863, 6864 & 6865, 6866 & 6867, 6868 & 6870, 6869 & 6870 and 253 & 3998 were used to amplify the MCH1, MCH2, MCH5, AQY1, ITR1 and PDR12 loci, respectively. The expected sizes of the PCR products obtained when the gene is present (left) or deleted (right) are indicated. (c) Example of a diagnostic gel from the transformation introducing six simultaneous gene deletions, resulting in IMX717. The first lane (L) contains the GeneRuler DNA Ladder Mix. Lane 1–6 show the PCR results of the reference strain CEN.PK113–7D (top) and a randomly picked colony of IMX717 (bottom) with primers 6862 & 6863 for MCH1 (lane 1), 6864 & 6865 for MCH2 (lane 2), 6866 & 6867 for MCH5 (lane 3), 6868 & 5593 for AQY1 (lane 4), 6869 & 6870 ITR1 (lane 5) and 253 & 3998 for PDR12 (lane 6).

Figure 4.
Figure 4.

Multiplexing CRISPR/Cas9 in S. cerevisiae. (a) Chromosomal integration of the six genes required for expression of a functional E. faecalis pyruvate dehydrogenase complex in the yeast cytosol. All six genes are flanked by 60 bp sequences enabling HR (indicated with black crosses). The first and the last fragments are homologous to 60 bp just up- and downstream of the ACS2 ORF, respectively, thus enabling repair of the Cas9-induced double strand break by HR (left panel). Deletion of ACS1 using a 120 bp dsDNA repair fragment is shown in the right panel. (b) Multiplex colony PCR was performed on 10 transformants to check their genotypes. Results are shown for two representative colonies, confirming the intended genotype. The PCR on the wild-type strain (CEN.PK113-7D) shows the predicted bands for the presence of the wild-type ACS1 and ACS2 alleles. Two DNA ladders were used; L1 refers to the GeneRuler DNA ladder (Thermo Scientific) and L2 to the GeneRuler 50 bp DNA ladder (Thermo Scientific). (The bands indicated with an asterisk reflect aspecific PCR products).

Figure 5.
Figure 5.

Simultaneous introduction of different single-nucleotide mutations in S. cerevisiae. (a) Transformation of IMX581 (ura3–52, can1Δ::CAS9) was performed with pUDR020, resulting in the introduction of two mutations in NAT1 and GET4. Underlined: the PAM sequences associated with the gRNA targets. In white: restriction sites present in the original gRNA targeting sequence (BamHI) and in the repair fragment used to correct the double-strand break (EcoRI). (b) Introduction of the double-strand break and subsequent repair using the mutagenic repair fragment resulted in a change of restriction site from BamHI to EcoRI. In this colony PCR example, primers 7030 & 7031 and 7036 & 7037 were used to amplify a part of the GET4 and NAT1 locus (lane 1 and 4, respectively) from CEN.PK113–7D and two colonies of IMX581, transformed with pUDR020 and mutagenic repair fragments. In lanes labelled 2 and 5: digestion of the PCR fragments with BamHI, which only results in digestion fragments of sizes 250 bp & 459 bp and 226 bp & 628 bp when the original restriction sites in GET4 and NAT1 are still present (CEN.PK113–7D). In lanes labelled 3 and 6: digestion of the PCR fragments with EcoRI, which only results in digestion fragments of sizes 259 bp & 450 bp and 227 bp & 627 bp when this new restriction site has been introduced via the mutagenic repair fragment (colony 1 and 2).

Similar articles

Cited by

References

    1. Annaluru N, Muller H, Mitchell LA, et al. Total synthesis of a functional designer eukaryotic chromosome. Science. 2014;344:55–8. - PMC - PubMed
    1. Bao Z, Xiao H, Liang J, et al. Homology-Integrated CRISPR − Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol. 2014 DOI: 10.1021/sb500255k. - PubMed
    1. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315:1709–12. - PubMed
    1. Beekwilder J, van Rossum HM, Koopman F, et al. Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production. J Biotechnol. 2014;192:383–92. - PubMed
    1. Botstein D, Fink GR. Yeast: an experimental organism for 21st century biology. Genetics. 2011;189:695–704. - PMC - PubMed

Publication types

MeSH terms

Substances