Fig 1.
CRISPR/Cas9 baculovirus mediated Cas9 expression in U-2 OS cells.
A) Schematic representation of the recombination of a pAceBac-Cas9 plasmid with a bacmid in EmBacY cells. The resulting bacmids were used for CRISPR/Cas9 baculovirus production in Sf9 cells. B) Representative FACS-profile showing GFP expression in U-2 OS cells treated with CRISPR/Cas9 baculovirus (MOI: 25). C) Western blot showing expression of Cas9 in U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). α-tubulin was used as a loading control.
Fig 2.
CRISPR/Cas9 baculovirus mediated knockout of CPC subunits in U-2 OS cells.
A) Western blot of CPC members from mitotic U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). α-tubulin was used as a loading control. B) Quantification of the Western blot shown in A. Protein levels were normalized over α-tubulin. C) Immunofluorescence images of mitotic U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25) were scored for loss of centromeric Aurora B. Bars represent the mean ± the standard deviation (SD) of 3 independent experiments. 100 cells were analyzed per experiment. D) Quantification of centromeric Aurora B levels in immunofluorescence images of mitotic U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). Aurora B levels were normalized over CENPC. Thirty cells were analyzed per condition. Immunofluorescence images of the indicated cells are shown. E) Mitotic progression of H2B-mCherry U-2 OS cells that were transduced with the indicated CRISPR/Cas9 baculovirus (MOI: 25) and control cells. The timing of each frame is indicated in minutes, with the first frame in prometaphase set to t = 0. The average percentage of cells displaying the depicted phenotype and the SD are indicated. The numbers represent the average of 2 experiments and at least 10 cells were analyzed per condition for each experiment. F) Time in mitosis for U-2 OS cells that were transduced with the indicated CRISPR/Cas9 baculovirus (MOI: 25) in the presence of 1 μM paclitaxel. Cells treated with 2 μM ZM447439 were used as a positive control for the override of a paclitaxel-induced mitotic delay. Each bar represents a single cell. At least 48 cells were analyzed for each condition. The color of the bar indicates cell fate as depicted in the figure legend.
Fig 3.
Effect of CRISPR/Cas9 baculoviral transduction in a panel of cell lines.
A) Representative FACS-profiles showing GFP expression in cells treated with Cas9-GFP baculovirus (MOI: 75). The markers are set such that 2% of the cells treated with Cas9-puro baculovirus are included in this region. The percentage of cells treated with Cas9-GFP baculovirus within the marker region is indicated. B) Immunofluorescence images of mitotic cells treated with the indicated Cas9-GFP baculoviruses (MOI: 75) were scored by eye for the presence or absence of centromeric Aurora B. The bars represent the mean ± SD of 2 experiments. At least 24 cells were analyzed per experiment per condition.
Fig 4.
Introduction of the H250Y mutation in Aurora B.
A) Schematic representation of the introduction of an HDR template carrying point mutations in the pAceBac-Cas9 plasmid. B) Colony formation of HCT116 cells in ZM447439 with cells carrying either wildtype Aurora B alleles, or alleles with homozygous H250Y mutations, or with a heterozygous mutation for H250Y combined with an indel in the other allele. The numbers indicate colony outgrowth in the ZM447439 treated conditions relative to the untreated conditions. Clones were also tested for their capacity to form colonies in the presence of puromycin. C) Sequence chromatograms of the Aurora B locus in the vicinity of H250. Depicted is the Aurora B sequence trace of wildtype HCT116 cells or clones carrying either the homozygous Aurora B H250Y mutation or the heterozygous Aurora B H250Y mutation combined with an indel. The PAM site is indicated and the arrows point out the base substitutions that were introduced. The corresponding amino acid sequence is shown in the top graph. D) Representative immunofluorescence images of prometaphase cells that have either retained or lost histone H3 Serine 10 (H3S10) phosphorylation after treatment with ZM447439 (0.5 μM). E) Quantification of immunofluorescence images of prometaphase cells depicted in (D) Cells were scored for H3S10ph loss following treatment with ZM447439 (0.5 μM). F) Immunofluorescence images of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75) were arrested in mitosis and scored for loss of centromeric Aurora B. G) Representative FACS-profiles showing the DNA content of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations that were treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75). Puromycin treatment was used to select for transduced cells and samples were harvested 4 days after transduction.
Fig 5.
Endogenous tagging of Haspin using CRISPR/Cas9 baculovirus.
A) Schematic representation of the introduction of an HDR template for endogenous tagging of GSG2 in the pAceBac-Cas9 plasmid. B) Integration of the YFP-tag at the C-terminus of the gene encoding Haspin was confirmed by PCR using the indicated primer pairs, schematically depicted in Fig 5A. The PCR product obtained using primers 1 and 2 is of the untagged allele, based on its size. C) Representative live cell images of U-2 OS-LacO Haspin-YFP cells and RPE-1 Haspin-YFP cells in prometaphase and interphase. D) Schematic overview depicting the binding of LacI-tagRFP-Pds5B to the LacO repeats. Upon interaction between Pds5B and Haspin an YFP signal can be detected at this ectopic locus. E) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B and stained for DAPI, YFP and RFP. F) Quantification of Haspin-YFP at the LacO locus. Depicted is the mean of Haspin-YFP normalized over RFP ± SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. G) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B stained for DAPI, H3T3ph and RFP. H) Quantifications of H3T3ph at the LacO locus. Depicted is the mean of H3T3ph levels normalized over RFP ± the SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. A minimum of 23 cells was analyzed per experiment.
Table 1.
List of sgRNA sequences used in this study.