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. 2020 Aug 28;11(1):4344.
doi: 10.1038/s41467-020-18222-0.

Chromosome drives via CRISPR-Cas9 in yeast

Hui Xu  1   2 , Mingzhe Han  1   2 , Shiyi Zhou  1   2 , Bing-Zhi Li  1   2 , Yi Wu  3   4 , Ying-Jin Yuan  1   2
Affiliations

Chromosome drives via CRISPR-Cas9 in yeast

Hui Xu et al. Nat Commun. .

Abstract

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Chromosome elimination by CRISPR–Cas9 induced one DSB near the centromere.
a Mechanism for chromosome elimination via CRISPR–Cas9. The gray chromosome represents chrIII, and the red chromosome represents synIII. The CEN represents the centromere and the mosaic red chromosome represents the process of chromosome elimination in the diploid strain. b All synthetic and native PCRtags on synIII were tested. 372 synthetic and wild-type PCRtags were used to verify chromosome elimination of synIII in yHX0295. c Southern blot analysis of synIII elimination. The genomes of the BY4741, synIII, diploid (BY4741-synIII) were used as controls and four synIII elimination diploid strains (yHX0295, yHX0333, yHX0334, and yHX0334) were analyzed. All genomes were digested with HindIII. ChrIII yielded a 10,650 bp fragment, and synIII yielded a 4857 bp fragment after digestion and hybridization with the probe. The blue frame indicates fragments from chrIII, and the orange frame indicates fragments from synIII. d Mating type test of CRISPR-containing diploid strains by tester A and tester α. SD solid media with tester A or tester α were mated to media with CRISPR-containing diploid strains. e Efficiency of chromosome elimination at different positions of synX. L-M represents the middle of the left arm, and L-T represents the telomere of the left arm. A total of 120 strains were tested for each replicate and the error bars indicate the standard deviations of all (n = 3) biological replicates. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Chromosome drives with a synthetic violacein pathway.
a Verification of chromosome endoreduplication via GFP and RFP in yeast. Strains were identified under visible light and observed for GFP and RFP. The haploid controls yHX0299 and yHX0141 displayed red and green color, respectively, under a fluorescence microscope. Three tetrads from the one driven strain, yHX0300, displayed a green color in all four spores. One tetrad was displayed at a higher resolution. One tetrad from the diploid control yHX0301 displayed two green spores and two red spores. Numbered squares indicate single tetrad shown at a higher resolution in the below panels. Scale bar, 10 µm. b Workflow of the chromosome-driven inheritance with components of the synthetic violacein pathway. c Whole-genome sequencing analysis of synX elimination in yHX0296. All the PCRTags in yHX0296 matched with the haploid control (yHX0186), and three of them were displayed. The red bases represent the mismatched bases of PCRTags compared yHX0296 with the haploid control (yHX0080). d The coverage map of yHX0296. The driven diploid strain yHX0296 was used for the analysis of chromosome endoreduplication. The sequencing depths of all sixteen chromosomes were at the same scale. The sequencing depth of chrX was similar to that of the other chromosomes. The red triangle indicates the position of chrX in the coverage map. e Phenotypic test of spores from dissected tetrads. The spores with the synthetic violacein pathway produce purple colonies on SC media. In the absence of Cas9, normal 2:2 segregation was observed. A total of 87.14% of dissected tetrads were observed as having four purple spores. The spores of all 70 dissected tetrads are shown in Supplementary Fig. 6. The red frame indicates four spores from one driven strain and the orange frame indicates four spores from one undriven strain. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Preferential transmission of complex genetic traits in yeast via chromosome drive.
a Process by which chromosome drive transmits synXII by mating and sporulation. SynXII is encoded by a, highly repetitive rDNA sequence of S. bayanus. b Sequences of PCR products amplified from ITS regions for BY4741, JDY476 and four driven spores. The red frame indicates the sequence difference in rDNA between S. cerevisiae and S. bayanus. c Restriction enzyme digestion to verify the absence of the native ITS sequence in the driven spores. The PCR products were treated with (bottom) or without (top) ApaI. Four driven spores (yHX0290, yHX0291, yHX0292, and yHX0293) was used for the test. BY4741 and JDY476 were used as negative and positive controls, respectively. d Process by which chromosome drive transmits thermotolerance-related chromosomes. The detailed process of the construction of the heterozygous haploid is shown in Supplementary Fig. 12. e Distribution of SNPs throughout the entire chrXIV for four driven spores (yHX0285, yHX0286, yHX0287, and yHX0288) and Y12. The number of SNPs was counted every 26 kb, with chrXIV of BY4741 used as a control. f Serial dilution assay of four driven spores (yHX0285, yHX0286, yHX0287, and yHX0288) at 30, 37, and 40 °C. BY4742 and yHX0266 were used as controls. g Process by which chromosome drive transmits SCRaMbLEd synX with an unresolved chromium resistance trait. h Electrophoretic karyotype analysis of four dissected spores (yHX0236, yHX0237, yHX238, and yHX0239) via PFGE. Strains BY4742, synX, and yHX0065 were used as controls for chrX, synX, and SCRaMbLEd synX, respectively. The purple triangle indicates the positions of chrX, the red triangle indicates the positions of synX and the yellow triangle indicates the positions of SCRaMbLEd synX. i Serial dilution assay of four driven spores (yHX0236, yHX0237, yHX238, and yHX0239) under 0.15 mM Cd(NO3)2 for 72 h. Strains synX and BY4742 were used as controls. Source data are provided as a Source Data file.
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