Article Figures & Data
Figures
- 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
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
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
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
- Table 1 Yeast strains
Strain Mating Genotype Source YZY558 h+ ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 This study YZY559 h+ ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 ade6-M210 This study YZY560 h+ ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 ade6-M216 This study YZY561 h− ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 This study YZY562 h− ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 ade6-M210 This study YZY563 h− ura4-D18 leu1-Δ0 his3-Δ0 lys9-Δ0 ade6-M216 This study BP231 h+ ura4-D18 Jürg Kohli BP232 h− ura4-D18 Jürg Kohli YZY579 h+ leu1-Δ0 This study YZY580 h− leu1-Δ0 This study YZY581 h+ his3-Δ0 This study YZY582 h− his3-Δ0 This study YZY583 h+ lys9-Δ0 This study YZY584 h− lys9-Δ0 This study YZY585 h+ ura4-D18 leu1-Δ0 This study YZY586 h− ura4-D18 leu1-Δ0 This study YZY587 h+ ura4-D18 his3-Δ0 This study YZY588 h− ura4-D18 his3-Δ0 This study YZY589 h+ ura4-D18 lys9-Δ0 This study YZY590 h− ura4-D18 lys9-Δ0 This study YZY591 h+ leu1-Δ0 his3-Δ0 This study YZY592 h− leu1-Δ0 his3-Δ0 This study YZY593 h+ leu1-Δ0 lys9-Δ0 This study YZY594 h− leu1-Δ0 lys9-Δ0 This study YZY595 h+ his3-Δ0 lys9-Δ0 This study YZY596 h− his3-Δ0 lys9-Δ0 This study YZY597 h+ ura4-D18 leu1-Δ0 his3-Δ0 This study YZY598 h− ura4-D18 leu1-Δ0 his3-Δ0 This study YZY599 h+ ura4-D18 leu1-Δ0 lys9-Δ0 This study YZY600 h− ura4-D18 leu1-Δ0 lys9-Δ0 This study YZY601 h+ ura4-D18 his3-Δ0 lys9-Δ0 This study YZY602 h− ura4-D18 his3-Δ0 lys9-Δ0 This study YZY603 h+ leu1-Δ0 his3-Δ0 lys9-Δ0 This study YZY604 h− leu1-Δ0 his3-Δ0 lys9-Δ0 This study - Table 2 Plasmids
Plasmida Antibiotic Marker Description Addgene ID pYZ033 AmpR Sp ura4 Entry vector for S. pombe CRISPR-Cas9 system. 98404 pYZ292 AmpR Sc LEU2 Entry vector for S. pombe CRISPR-Cas9 system. 102686 pYZ293 AmpR Sp leu1 Entry vector for S. pombe CRISPR-Cas9 system. 102687 pYZ294 AmpR kanMX Entry vector for S. pombe CRISPR-Cas9 system. 102688 pYZ300 AmpR Sp his3 Entry vector for S. pombe CRISPR-Cas9 system. 102689 pYZ301 AmpR Sp lys9 Entry vector for S. pombe CRISPR-Cas9 system. 102690 pYZ145 AmpR — Donor DNA for leu1 deletion in S. pombe 98405 pYZ146 AmpR Sp ura4 Cas9-gRNA plasmid for leu1 98406 pYZ149 AmpR — Donor DNA for his3 deletion in S. pombe 98407 pYZ164 AmpR Sp ura4 Cas9-gRNA plasmid for his3 98408 pYZ172 AmpR — Donor DNA for lys9 deletion in S. pombe 98409 pYZ173 AmpR Sp ura4 Cas9-gRNA plasmid for lys9 98410 pYZ155 AmpR Sp ura4 nmt1 promoter: expression vector in S. pombe 98411 pYZ156 AmpR Sp ura4 nmt41 promoter: expression vector in S. pombe 98412 pYZ157 AmpR Sp ura4 nmt81 promoter: expression vector in S. pombe 98413 pYZ190 AmpR Sp ura4 General purpose vector in S. pombe 98428
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)
- Figure S1 -
