Comparison of mutation type and and cbPCR results.

<p>Comparison of mutation type and and cbPCR results.</p

Refinement of cbPCR.

<p>(A) PCR of Plpp2 locus was performed using the indicated primer sets. When F-out was absent, Plpp2 inner amplicon was detected in wild type DNA (lane 1), but was barely detected when doing cbPCR (lane 3). (B) Decreasing F-out primer length enabled detection of inner amplicons in cbPCR using wild type DNA and discrimination of mutant gDNA. (C) Using the optimized primers, completely mutant gDNA was discriminated from wild type or heterozygous mutant gDNA. Bands with expected sizes are marked with dots. The other bands were ignored as being either nonspecific or heteroduplexes. The calculated ratios between out- and in-amplicons are written below each lane. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179165#pone.0179165.s001" target="_blank">S1 Fig</a> for quantification of signals. WT or W: wild type, H: heterozygous, KO or K: knockout (mutant).</p

Optimization of cbPCR.

<p>(A) Nomenclature of the primers based on their orientation, using Sgpl1 locus as an example. The Cas9 cleavage site is zoomed in to show where the inner primers bind. (B) PCR was performed with or without the addition of the indicated inner primer. The ratios between out- and in-amplicons were calculated. The orientation of the inner primer affected the degree of competition between amplicons. See the changes in the 761 bp- or 748 bp-outer amplicons. The same experiment is performed in two different loci, namely Sgpl1 and Sphk1. (C) PCR was performed with the addition of inner primers of different lengths. The ratios between out- and in-amplicons were calculated. The length of the inner primer affected the degree of competition between amplicons (as seen by the decrease of the 761 bp-outer amplicon) and the tolerance to the mutations (as seen by the appearance of the ~531 bp-inner amplicon when using mutant DNA). (D) Mutants can be discriminated by cbPCR in most of the targets with a common primer design. Bands with expected sizes are marked with dots. The other bands were ignored as being either nonspecific or heteroduplexes. WT or W: wild type, KO or K: knockout (mutant).</p

Competition between amplicons enables the discrimination of mutant cells.

<p>(A) Design of the PCR primers to test whether Sgpl1-mutant cells can be discriminated by conventional endpoint PCR. The 3' end of the forward primer is zoomed to show its location relative to the Cas9 cleavage site. (B) Conventional endpoint PCR with the primer design shown in (A) was performed using wild type or mutant gDNA. The levels of PCR products did not change depending on genotypes or template DNA amount. (C) Design of competition-based PCR (cbPCR) and nomenclature of primers and amplicons. F-in and R-out are the same primers as forward and reverse primers in (A), respectively. (D) Basis of competition between amplicons that occur during cbPCR. (E) PCR was performed with the indicated primer sets, using gDNA from wild type or Sgpl1-mutant cells. The pattern of competition between cbPCR amplicons differed between wild type and mutant gDNA. (F) In contrast to conventional endpoint PCR, cbPCR enables the discrimination of wild type and mutant gDNA. The ratios between out- and in-amplicons are indicated. WT: wild type, KO: knockout (mutant).</p

Accuracy of cbPCR to detect mutants.

<p>(A) Strategy to compare the efficacy of mutant detection by cbPCR. (B) Survival of different clones after HPRT1 editing and 6-TG selection. Viable cells were stained with crystal violet. Clone 7 is absent due to a growth arrest before experiments. (C) Result of cbPCR in the clones of (B). The bands of expected sizes are illustrated by arrowheads. (D) Bands of (C) were quantified and ratios between out- and in-amplicons were calculated. Ranking based on the calculated ratios discriminated 6-TG resistant clones, except for the 6-TG sensitive clone #10. (E) Clone 10 had a point mutation. (F) Sanger sequencing of clone 10 revealed that the point mutation was silent. The other allele had a +1 insertion. Mutations are illustrated in magenta.</p

CPY secretion and Sna3-GFP trafficking and invertase activity are the same in <i>ret1-1 rsp5-1</i> mutant and in single <i>rsp5-1</i>.

<p>(<b>A</b>) Double mutants <i>ret1-1 rsp5-1</i> and <i>sec27-1 rsp5-1</i> do not secrete more CPY compared with the single <i>ret1-1</i>, <i>sec27-1</i> or <i>rsp5-1</i> mutants. Spore clones (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a>) were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting CPY were identified by Western blotting with anti-CPY antibody. (<b>B</b>) Sna3-GFP trafficking defect caused by <i>rsp5-1</i> mutation is not augmented by <i>ret1-1</i>. Plasmid encoding Sna3-GFP was transformed into spore clones as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2A</a>. Transformants were grown to mid logarithmic phase on SC -ura at 30°C or shifted to 35°C for 1 hour. Sna3-GFP was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole and viewed with Nomarski optics (NOM). (<b>C</b>) Whole cell lysates form transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g003" target="_blank">Figure 3B</a> were analyzed by Western blotting with anti-GFP antibody. Percentage of Sna3-GFP in total GFP signal (GFP and Sna3-GFP) is given. (<b>D</b>) Invertase activity was assayed in spore clones as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2A</a>. Cultures were grown to mid logarithmic phase at 30°C and shifted or not to 35°C for 30 minutes. The proportion between activity of secreted invertase to the total invertase activity (invertase secretion index) is shown.</p

<i>rsp5</i> and <i>bre5</i> mutations have additive effect on growth and mislocalization of GFP-Rer1.

<p>(<b>A</b>) <i>bre5Δ</i> and <i>HA-rsp5-1</i> mutations show weak genetic interaction. Strains <i>doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1</i> and <i>doa4Δ bre5Δ HA-rsp5-1</i> were transformed with plasmid encoding <i>DOA4</i>. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (<b>B</b>) <i>doa4Δ bre5Δ HA-rsp5-1</i> mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.</p

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Overexpression of ubiquitin suppresses <i>ret1-1 rsp5-1</i> mutant defects.

<p>(<b>A</b>) Growth defect of <i>ret1-1 rsp5-1</i> mutant is suppressed by overexpression of ubiquitin or its variants. <i>ret1-1 rsp5-1</i> mutant was transformed with empty vector [-] or with plasmids encoding wild type ubiquitin [UBI], ubiquitin with only single lysine 48 [K48] or 63 [K63] present and all other lysines replaced with arginine. Serial 1∶10 dilutions of transformants were spotted on YPD medium and incubated for 2 days at indicated temperatures. (<b>B</b>) Localization of GFP-Rer1 to vacuole in <i>ret1-1 rsp5-1</i> mutant is suppressed by overexpression of ubiquitin. Transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6A</a> were additionally transformed with plasmid encoding GFP-Rer1 and were grown on SC -ura -leu at 28°C. Expression of ubiquitin variants was induced by addition of 100 µM CuSO₄ for 2 hours before observations. GFP-Rer1 was localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>C</b>) Whole cell lysates form transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6B</a> were analyzed by Western blotting with anti-GFP antibody. Percentage of GFP-Rer1 in total GFP signal (GFP and GFP-Rer1) in each lane is given. (<b>D</b>) Wild type or <i>rsp5-1</i> mutant were transformed with a plasmid expressing <i>His₆-UBI</i> and with empty vector [-] or a plasmid encoding GFP-Rer1. Transfromants were grown to mid-logarithmic phase at 30°C. Expression of ubiquitin was induced by addition of 100 µM CuSO₄ for 2 hours before cultures were shifted or not to 37°C for 1 hour. His₆-Ubi was pulled down on Ni-NTA beads. Total fraction and fraction bound to beads were analysed by Western blotting with anti-GFP antibody. (<b>E</b>) Fusion protein Ub-GFP-Rer1 is targeted to vacuole. The <i>rer1-1 rsp5-1</i> strain was transformed with plasmid encoding one of the fusions <i>GFP-RER1</i> or <i>Ub-GFP-RER1</i>. The GFP-Rer1 and Ub-GFP-Rer1 proteins were localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>F</b>) Ub-GFP-Rer1 protein is expressed. Total protein extracts from the same transformants as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6E</a> were analyzed by Western blotting with anti-GFP antibody.</p

Retrograde trafficking from Golgi to ER is impaired in double <i>ret1-1 rsp5-1</i> mutant.

<p>(<b>A</b>) Double mutant <i>ret1-1 rsp5-1</i> is sensitive to neomycin. Spore clones from <i>ret1-1</i> × <i>rsp5-1</i> cross (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a>) were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (<b>B</b> and <b>C</b>) Defect of GFP-Rer1 trafficking in <i>ret1-1 rsp5-1</i> and <i>sec27-1 rsp5-1</i> mutants. Spore clones (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g003" target="_blank">3B</a>) from <i>ret1-1</i> × <i>rsp5-1</i> and <i>sec27-1</i> × <i>rsp5-1</i> crosses were transformed with plasmid encoding <i>GFP-RER1</i>. Transformants were grown on SC -ura at 28°C. GFP-Rer1 was localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>D</b>) <i>rsp5-1</i> mutation is responsible for additional defect in GFP-Rer1 trafficking caused by <i>ret1-1</i>. Centromeric vector encoding <i>HA-RSP5</i> or empty vector ([-]) were transformed into <i>ret1-1 rsp5-1</i> expressing <i>GFP-RER1</i>. Whole cell protein extracts from transformants were analysed by Western blotting with anti-GFP antibody. (<b>E</b>) Secretion of Kar2p is enhanced in <i>ret1-1 rsp5-1</i> mutant. Spore clones from <i>ret1-1</i> × <i>rsp5-1</i> cross (as in B) were grown at 28°C in YPD, transferred to fresh medium and incubated at 28°C for 1 h. Whole cell protein extracts and proteins TCA-precipitated from medium were analyzed by Western blotting with anti-Kar2 and anti-PGK antibody. The latter was to control cell integrity. (<b>F</b>) HA-Rsp5 binds COPI complex. The extracts from <i>rsp5Δ</i> strain transformed with empty vector ([-]) or with centromeric plasmid YCpHA-RSP5 ([<i>HA-RSP5</i>]) were used for immunoprecipitation using anti-HA antibody (16B12). Total extracts (T) and immunoprecipitated fraction (IP) were analysed by Western blotting with anti-HA and with anti-coatomer antibody.</p