Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

Abstract

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Keywords: EST3; Saccharomyces cerevisiae; centromere destabilization; chromoduction; chromosome debugging; chromosome substitution; megachunk assembly; synIX; transcriptomics.

Graphical abstract

Figure 1

Design and assembly of synIX (A) Diagram of the differences between wild-type (WT) chrIX and synIX. Designer features specific to synIX include addition of PCRTags and loxPsym sites, recoding of TAG stop codons to TAA, replacement of WT telomeres with universal telomere caps (UTCs), and removal of tRNA genes (relocated to a tRNA neochromosome), introns, retrotransposons, and subtelomeric repeats. (B) Schematic of the synIXL construction process, reflecting synthetic and (unwanted) WT composition of chrIX in the synIX strain after each stage of assembly. Stage 1: SwAP-In with minichunks (A–F). Stage 2: SwAP-In with preassembled megachunks (G–I, opaque with blue lines). Stage 3: error correction with CRISPR-Cas9 (primarily in highlighted megachunks A–F). Blue: syn minichunks, red: WT minichunks, gray: megachunks. Red patches in stages 1 and 2 reflect segments of DNA that were not replaced by their expected synthetic minichunk counterparts during SwAP-In with minichunks in stage 1 (A–F); appropriate synthetic sequences were integrated at these sites during stage 3. kb, kilobase pairs. (C) Minichunk integration strategy for assembling megachunks A–F of synIX in yeast (used for stage 1 in B). KanMX marker (black) was integrated into left end of WT chrIX (red). Individual minichunks (blue, synthetic) comprising one megachunk were then co-transformed into in-progress synIX strain and used to overwrite WT chrIX sequence (red) via homologous recombination. Alternating auxotrophic markers (LEU2, brown, and URA3, orange) were used and overwritten at each step. (D) Megachunk plasmid assembly and integration approach for building and integrating megachunks G–I in synIX (used for stage 2 in B). Minichunks (blue) were assembled in yeast, and an auxotrophic marker (here, URA3, orange) was added to the end of each assembly, followed by a segment of chrIX homology. Megachunk assembly overwrote prior round auxotrophic marker (brown, LEU2) and WT DNA (red) by homologous recombination. (E) Schematic depicting frameshift mutation in FAA3 gene in synIX design. Synthetic sequence included 7 bp not present in S288C WT yeast reference genome. Added bases (red) cause a frameshift, with resulting synIX amino acid sequence (red) varying from the expected chrIX amino acid sequence (black, bottom). (F) Coverage plots showing read depth along each yeast chromosome in disomic synIX strain yLHM0588 (top) and monosomic synIX strain yLHM0721 (bottom). The x axis: yeast chromosome (not to scale). The y axis: relative depth based on number of reads at each position divided by average read depth across sixteen yeast chromosomes. See also Figures S1–S3 and Tables S2, S3–S5, and S8.

Figure 2

Identifying and mapping an EST3-related bug (A) Spot assays comparing the growth of BY4741 (WT) to yLHM1192 (synthetic) across a variety of growth conditions. Each column represents a 10-fold dilution. RT, room temperature, ∼22°C. (B) (Top) Schematic of region near CEN9 identified as containing gene responsible for synIX fitness defect. Unhealthy synIX strain (yLHM1192) was transformed with plasmids containing transcription units for each gene shown. (Bottom) Spot assays comparing growth of BY4741 and yLHM1192 to yLHM1192 transformed with plasmid containing EST3 coding sequence plus 500 bp upstream sequence and 200 bp downstream sequence. Each column represents a 10-fold dilution. (C) Spot assays comparing growth of strains with different combinations of synthetic and WT features in and upstream of EST3. Each column represents a 10-fold dilution. Schematics on the left illustrate WT (red) and synthetic (blue) features present in each strain. Asterisk (∗) marks site of known EST3 programmed +1 ribosomal frameshift. See also Figures S4–S7.

Figure 3

Modifying synIX strains upstream of EST3 alters fitness and telomere length (A) Spot assays and accompanying schematics of the region upstream of EST3 for strains BY4741 (WT chrIX), yLHM1192 (synthetic), and variant strains derived from yLHM1192. WT PCRTags, tRNA^Asp gene, and Ty1 LTR, red; synthetic PCRTags and loxPsym sites, blue; mutation in tRNA^Asp gene, vertical black line. Each spot assay column represents a 10-fold dilution. (B) Southern blot of XhoI-digested yeast gDNA derived from BY4741 (WT) and replicate strains with modifications equivalent to yLHM1192 (synthetic) and variant strains (yLHM1504, yLHM1505, yLHM1506, yLHM1591, and yLHM1601, as depicted in A). DNA was probed with a digoxigenin-labeled fragment specific for telomeric repeats. Black triangle (right): average size of Y′-containing telomeric fragments for BY4741. Gray triangle (right): average size of Y′-containing telomeric fragments for yLHM1192. Left and middle: left and right halves of one southern blot, cropped to remove one lane in the middle. Right: right side of second southern blot, cropped to remove left-hand ladder, BY4741, and yLHM1192 lanes. See also Figure S8 and Table S6.

Figure 4

Modifying WT yeast strains upstream of EST3 alters telomere length (A) Schematics of region upstream of EST3 for strains BY4741 (WT chrIX), yLHM1192-equivalent (synthetic modification matching yLHM1192 in otherwise WT yeast strain), and variant strains derived from the yLHM1192-equivalent strain. WT PCRTags, tRNA^Asp gene, and Ty1 LTR, red; loxPsym sites, blue; mutation in tRNA^Asp gene, vertical black line. (B) Southern blot of XhoI-digested yeast gDNA derived from BY4741 (WT), yLHM1192 (synthetic), and replicate strains with synthetic-equivalent modifications made upstream of EST3, as depicted in (A). DNA was probed with a digoxigenin-labeled fragment specific for telomeric repeats. Black triangle (right): average size of Y′-containing telomeric fragments for BY4741. Gray triangle (right): average size of Y′-containing telomeric fragments for BY4741 + yLHM1192-equivalent modification strain. See also Figure S9 and Table S6.

Figure 5

Dissecting effects of EST3-adjacent features on Est3 protein and EST3 RNA expression (A) Immunoblotting immunoprecipitated (IP/IB) Est3p (top) and H3 histone pre-IP loading control (bottom). Est3p was C-terminally tagged with 3× FLAG in each synthetic strain, and diploids were generated via mating with WT BY4742. Two independent colonies were grown in YPD at 37°C for each strain. Right-most lane (second ladder) was cropped out of image. (B) IP/IB Est3p (top) and H3 histone pre-IP loading control (bottom) for low-expressing Est3p strains plus controls, as in (A), except that three independent colonies were grown for each yLHM1192-, yLHM1505-, and yLHM1591-derived diploid strain. (C) Strand-specific RNA sequencing alignments for the reverse strand of BY4741 and yLHM1192 (strand from which EST3 transcription is expected, i.e., “reverse-strand reads”) grown at 37°C in YPD to a composite reference containing both synthetic and native IX features. Schematic illustrates WT chrIX (red) and synthetic (blue) features. Black box on RNA sequencing alignment plot highlights region between expected EST3 start codon and upstream tRNA. Asterisk (∗) marks site of known EST3 programmed +1 ribosomal frameshift. (D) Quantification of reverse-strand reads mapping directly upstream of EST3 (between tRNA and start codon, corresponding to black box in C) as a fraction of total reverse-strand EST3 coding and upstream reads for each strain shown in (C). Read counts were based on alignment to native (BY4741) or synthetic (yLHM1192) reference sequences. Error bars represent SD of three biological replicates. (E) Spot assays and strand-specific RNA sequencing alignments for reverse-strand reads aligning to EST3 region for samples grown at 37°C in YPD. WT PCRTags, tRNA^Asp gene, and Ty1 LTR, red; synthetic PCRTags and loxPsym sites, blue; mutation in tRNA^Asp gene, vertical black line. Asterisk (∗) marks site of known EST3 programmed +1 ribosomal frameshift. (F) Quantification of reverse-strand reads mapping directly upstream of EST3 (between tRNA and start codon, corresponding to black box in E) as fraction of total reverse-strand EST3 coding and upstream reads for each strain shown in (E). Read counts were based on alignment to native (BY4741) or synthetic (yLHM1192) reference sequences. Error bars represent SD of three biological replicates. (G) Nanopore direct RNA sequencing reads aligned to EST3 region (±3 kb). The x axis: chromosome coordinate. The y axis: number of reads. Forward-strand reads are above the y axis, and reverse-strand reads are below the y axis. Dotted lines indicate boundaries of EST3 coding region. Yellow reads: “nontranslatable” reads containing at least one AUG upstream of expected EST3 start codon plus a minimum of 20 bp 5′ UTR sequence. Blue reads: reads mapping to EST3 that start no more than 20 bp upstream of first AUG upstream of expected EST3 start codon, including “translatable/functional” reads spanning the entirety of EST3 and reads starting downstream of EST3-initiating AUG. See also Figures S10–S16 and Tables S6–S7.

Figure 6

SynIX characterization (A) Spot assays comparing growth of BY4741 (WT) to yLHM1601 (synthetic) across several conditions. Each column represents a 10-fold dilution. RT, room temperature, ∼22°C. (B) DNA sequencing coverage plot for synIX strain (yLHM1601). The x axis: yeast chromosome (not to scale) or coordinate along synIX. Relative depth (y axis) based on reads mapped to yeast_chr09_3_55 reference sequence divided by average read depth across sixteen yeast chromosomes (top) or synIX (bottom). (C) Pulsed-field gel showing chromosomes from BY4741 (chrIX), yLHM1601 (synIX), yLHM2337 (BY4742-background strain with copies of chrIX and synIX), and yLHM2401 (yLHM2337 after loss of chrIX) strains. Vertical black line indicates location of potential discrepancy in band intensity for larger chromosomes between BY4741 and yLHM1601; synIX was moved to an alternative strain (yLHM2337) with larger bands resembling BY4741 in intensity, and chrIX was subsequently lost from this strain (producing yLHM2401) via strategy described in next section. (D and E) Volcano plots of differentially expressed genes obtained from RNA sequencing data for synIX strain (yLHM1601) vs. BY4741 measured at 30°C (D) and 37°C (E) in YPD. Upregulated genes in yLHM1601 depicted in red (chrIX) and downregulated genes in medium blue (chrIX) or light blue (other chromosomes). Transcript counts based on alignment to S288C reference transcriptome. The auxotrophic gene LYS2 is present in BY4741 but not in yLHM1601. Fold change cutoff is 4, and adjusted p value cutoff is 0.01. Three biological replicates were used for each strain. For corresponding unfiltered plots of RNA sequencing data depicting all measured genes, see Figures S20A and S20B. See also Figures S17–S19 and Table S8.

Figure 7

Chromosome substitution of synIX (A) Chromosome substitution occurs in two steps, chromoduction, in which synIX is transferred to recipient strain, followed by counterselection against WT chrIX to remove native version of chromosome and restore normal chromosome copy number in chromoductant strain. Schematic diagram of substitution steps. Black bars, WT chromosomes. Blue bars, synIX. Recipient strain harbors an ade2 mutation and thus forms pink colonies for easily distinguishing chromoductants. (B) PFGE of donor strain, recipient strain, disomic chromoductants, and chromoductants after counterselection against chrIX. BY4741 serves as control. chrIX and synIX chromosomes indicated with black arrows. Green triangles indicate synIX in disomic chromoductants. Red triangles indicate extra chrII or chrIII in disomic chromoductants. (C) Whole-genome sequencing read depth plots of disomic chromoductants (yWZ610, yWZ612) and chrIX counterselected (i.e., fully substituted) chromoductants (yWZ618). The x axis: sixteen yeast chromosomes, not drawn to scale. The y axis: relative read depth, calculated as number of reads at each position divided by average read depth across sixteen yeast chromosomes. (D) chrII SNPs in chromoductant strains before and after counterselection against chrIX. Recipient strain SNP: orange, donor strain SNP: blue. Strains: yWZ610: chromoductant strain with one copy of chrII from donor strain and one from recipient strain (mixed SNP population); yWZ611: chromoductant strain with two copies of chrII from recipient strain (endoreduplicated chrII); yWZ618: strain derived from yWZ610 after counterselection against chrIX, with one copy of chrII harboring recipient strain SNP; yWZ619: strain derived from yWZ611 after counterselection against chrIX, with one copy of chrII harboring recipient strain SNP. (E) Relative depth plot for chromosome substitution strains prior to counterselection against chrIX. The x axis: sixteen yeast chromosomes, not drawn to scale. The y axis: relative read depth. (F) Relative depth plot for chromosome substitution strains after counterselection against chrIX. The x axis: sixteen yeast chromosomes, not drawn to scale. The y axis: relative read depth. See also Figure S21 and Table S9.