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Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors

View ORCID ProfileYu Zhao and View ORCID ProfileJef D. Boeke
G3: Genes, Genomes, Genetics March 1, 2018 vol. 8 no. 3 789-796; https://doi.org/10.1534/g3.117.300363
Yu Zhao
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
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  • ORCID record for Yu Zhao
Jef D. Boeke
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
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  • Figure 1
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    Figure 1

    Design of CRISPR-Cas9 toolbox and leu1-Δ0 in S. pombe. (A) ura4-D18 structure. The ura4+ transcript is shown in purple. The tam14+ and new25+ transcripts are shown in gray. (B) For each marker, one 20 bp gRNA sequence was selected, followed by the PAM sequence NGG. (C) Toolbox consists of a Cas9-gRNA expression vector specified for each deletion and another NotI-linearized donor DNA. (D) leu1-Δ0 deletion. A 1.9 kb region between the apc10+ and top2+ transcript ends was deleted. The leu1-32 mutation site was labeled in purple. The gRNA targeting leu1 was selected from the leu1+ CDS and labeled in red. (E) To minimize the effect on cell growth, one 119-bp AT-rich terminator (Scirica et al. 2013) from chromosome I was integrated. This design was achieved through the donor DNA in pYZ145. AmpR, ampicillin resistance; CDS, coding sequence; CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA; HR, homologous recombination; PAM, protospacer adjacent motif; UTR, untranslated region.

  • Figure 2
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    Figure 2

    Design of deletions and donor DNAs for his3-Δ0 and lys9-Δ0. (A) A 2.0 kb region containing the his3+ CDS was deleted, generating his3-Δ0. The gRNA was selected upstream of the CDS in order to make the toolbox compatible with existing his3-D1 strains. The region 200 bp upstream of the 5′ UTR was arbitrarily defined as the putative promoter in the plasmid. (B) The his3+ gene has three introns and four exons. The convergently transcribed downstream gene alg2+ is essential, and its transcript ends in the second exon of his3+. The poly(A) signal located at the second intron, and the first intron, may work as the terminator for alg2+. (C) The region from the first to second introns was kept in the genome for his3-Δ0, in order to minimize any possible effects on alg2+ transcripts. Since all the introns were removed when his3 was used as the marker, this design leaves no significant homologous region with the plasmid. The donor DNA was the NotI digestion product from the pYZ149 plasmid, and a 1 kb homologous region was included on each side for integration. (D) lys9 is a new selectable marker. The lys9-Δ0 strain is auxotrophic for lysine and the deletion has no detectable effect on cell growth. A 2.2 kb region was deleted, including the 1.6 kb transcription unit, and the 400 bp upstream and 150 bp downstream regions. The gRNA targets the CDS. Donor DNA is a NotI digest of pYZ172. AmpR, ampicillin resistance; CDS, coding sequence; gRNA, guide RNA; HR, homologous recombination; UTR, untranslated region.

  • Figure 3
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    Figure 3

    Deletion confirmation with replica plates and strain fitness assay. (A) From single colonies, we streaked the marker deletion strains on the YES plate. Then, the plate was replica plated to a PMG5 plate with all five supplements, and the indicated drop-out plates. (B) Fitness testing. Dots represent 10-fold dilutions left to right. Compared to wild-type or ura4-D18 strains, the new deletions caused no detectable growth defect. PMG5, S. pombe minimal glutamate medium with 5 supplements; YES, yeast extract with supplements.

  • Figure 4
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    Figure 4

    New vector designs for expression and general-purpose vectors. They all have four basic components: (1) an ampicillin resistance gene (AmpR), (2) an ars1 replication site for S. pombe, (3) a pUC/ColE1 replication region for E. coli, and (4) the lacZα multiple cloning site. (A) The expression vector diagram. In addition to the four basic parts, the empty BsaI-pad or one selectable marker is included. The marker gene contains several synonymous mutations to eliminate the restriction cutting sites. (B) The vector diagram for general purpose, e.g., complementation cloning. The nmt expression cassette was removed to minimize plasmid size. (C) In the lacZα multiple cloning site (MCS) of expression vectors, several unique cutting sites are available. This region is compatible with blue–white screening and can be sequenced with universal primers. (D) Five unique restriction enzyme cutting sites are designed in a second multiple cloning site downstream of the nmt promoter, which is consistent with the design of the pREP1/2 plasmid. These sites have been eliminated from the lacZα MCS region through synonymous mutations. (E) In the general vectors, original lacZα was used here with more unique restriction enzyme cutting sites. All sites indicated were checked for uniqueness.

Tables

  • Figures
  • Additional Files
  • Table 1 Yeast strains
    StrainMatingGenotypeSource
    YZY558h+ura4-D18leu1-Δ0his3-Δ0lys9-Δ0This study
    YZY559h+ura4-D18leu1-Δ0his3-Δ0lys9-Δ0ade6-M210This study
    YZY560h+ura4-D18leu1-Δ0his3-Δ0lys9-Δ0ade6-M216This study
    YZY561h−ura4-D18leu1-Δ0his3-Δ0lys9-Δ0This study
    YZY562h−ura4-D18leu1-Δ0his3-Δ0lys9-Δ0ade6-M210This study
    YZY563h−ura4-D18leu1-Δ0his3-Δ0lys9-Δ0ade6-M216This study
    BP231h+ura4-D18Jürg Kohli
    BP232h−ura4-D18Jürg Kohli
    YZY579h+leu1-Δ0This study
    YZY580h−leu1-Δ0This study
    YZY581h+his3-Δ0This study
    YZY582h−his3-Δ0This study
    YZY583h+lys9-Δ0This study
    YZY584h−lys9-Δ0This study
    YZY585h+ura4-D18leu1-Δ0This study
    YZY586h−ura4-D18leu1-Δ0This study
    YZY587h+ura4-D18his3-Δ0This study
    YZY588h−ura4-D18his3-Δ0This study
    YZY589h+ura4-D18lys9-Δ0This study
    YZY590h−ura4-D18lys9-Δ0This study
    YZY591h+leu1-Δ0his3-Δ0This study
    YZY592h−leu1-Δ0his3-Δ0This study
    YZY593h+leu1-Δ0lys9-Δ0This study
    YZY594h−leu1-Δ0lys9-Δ0This study
    YZY595h+his3-Δ0lys9-Δ0This study
    YZY596h−his3-Δ0lys9-Δ0This study
    YZY597h+ura4-D18leu1-Δ0his3-Δ0This study
    YZY598h−ura4-D18leu1-Δ0his3-Δ0This study
    YZY599h+ura4-D18leu1-Δ0lys9-Δ0This study
    YZY600h−ura4-D18leu1-Δ0lys9-Δ0This study
    YZY601h+ura4-D18his3-Δ0lys9-Δ0This study
    YZY602h−ura4-D18his3-Δ0lys9-Δ0This study
    YZY603h+leu1-Δ0his3-Δ0lys9-Δ0This study
    YZY604h−leu1-Δ0his3-Δ0lys9-Δ0This study
  • Table 2 Plasmids
    PlasmidaAntibioticMarkerDescriptionAddgene ID
    pYZ033AmpRSp ura4Entry vector for S. pombe CRISPR-Cas9 system.98404
    pYZ292AmpRSc LEU2Entry vector for S. pombe CRISPR-Cas9 system.102686
    pYZ293AmpRSp leu1Entry vector for S. pombe CRISPR-Cas9 system.102687
    pYZ294AmpRkanMXEntry vector for S. pombe CRISPR-Cas9 system.102688
    pYZ300AmpRSp his3Entry vector for S. pombe CRISPR-Cas9 system.102689
    pYZ301AmpRSp lys9Entry vector for S. pombe CRISPR-Cas9 system.102690
    pYZ145AmpR—Donor DNA for leu1 deletion in S. pombe98405
    pYZ146AmpRSp ura4Cas9-gRNA plasmid for leu198406
    pYZ149AmpR—Donor DNA for his3 deletion in S. pombe98407
    pYZ164AmpRSp ura4Cas9-gRNA plasmid for his398408
    pYZ172AmpR—Donor DNA for lys9 deletion in S. pombe98409
    pYZ173AmpRSp ura4Cas9-gRNA plasmid for lys998410
    pYZ155AmpRSp ura4nmt1 promoter: expression vector in S. pombe98411
    pYZ156AmpRSp ura4nmt41 promoter: expression vector in S. pombe98412
    pYZ157AmpRSp ura4nmt81 promoter: expression vector in S. pombe98413
    pYZ190AmpRSp ura4General purpose vector in S. pombe98428
    • ID, identifier; AmpR, ampicillin; CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA.

    • ↵a Only the newly designed plasmids with the Sp ura4 marker are shown here. Additional plasmids with BsaI-pad, leu1, his3, and lys9 are described in Table S2.

Additional Files

  • Figures
  • Tables
  • Supplemental Material for Zhao and Boeke, 2018

    • Figure S1 -

      Maker genes used in the plasmids to complement deletions in the genome. (.pdf)



    • Figure S2 -

      Transformation using newly designed plasmids into the auxotrophic strains.  (.jpg)



    • Table S1 -

      Newly constructed plasmid to introduce the selectable marker deletions in Sch. pombe. (.docx)

    • Table S2 -

      Plasmids newly designed and constructed in this study for general purpose or expression in Sch. pombe. (.docx)

    • Table S3 -

      Newly designed CRISPR/Cas9 plasmids used in Sch. pombe. (.docx)

    • Table S4 -

      Additional yeast strains. (.docx)

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Volume 8 Issue 3, March 2018

G3: Genes|Genomes|Genetics: 8 (3)

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Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors

View ORCID ProfileYu Zhao and View ORCID ProfileJef D. Boeke
G3: Genes, Genomes, Genetics March 1, 2018 vol. 8 no. 3 789-796; https://doi.org/10.1534/g3.117.300363
Yu Zhao
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Yu Zhao
Jef D. Boeke
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jef D. Boeke
  • For correspondence: jef.boeke@nyumc.org
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Citation

Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors

View ORCID ProfileYu Zhao and View ORCID ProfileJef D. Boeke
G3: Genes, Genomes, Genetics March 1, 2018 vol. 8 no. 3 789-796; https://doi.org/10.1534/g3.117.300363
Yu Zhao
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Yu Zhao
Jef D. Boeke
Institute for Systems Genetics, NYU Langone Health, New York 10016Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jef D. Boeke
  • For correspondence: jef.boeke@nyumc.org

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