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Reshuffling yeast chromosomes with CRISPR/Cas9
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Reshuffling yeast chromosomes with CRISPR/Cas9
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Paper
Reshuffling yeast chromosomes with CRISPR/Cas9
2018,2019
Overview
Untangling the phenotypic impact of chromosomal rearrangements from the contribution of the genetic background requires versatile procedures to generate structural variations. We developed a CRISPR/Cas9-based method to efficiently reshuffle the yeast genome in a scarless and markerless manner. Simultaneously generating two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs resulted reciprocal translocations at the base-pair resolution. We made these translocations either irreversible by deleting a small sequence at the junction or reversible to the original chromosomal configuration by inducing the backward translocation. Furthermore, generating multiple DSBs by targeting repeated sequences and using uncut copies of the repeats as template for trans-chromosomal repair resulted in a large diversity of karyotypes comprising multiple rearrangements including balanced and unbalanced variations. We validated the targeted translocations and characterized multiple rearrangements by long-read de novo genome assemblies. To test the phenotypic impact of rearranged chromosomes we first recapitulated in a lab strain the SSU1/ECM34 translocation believed to provide increased sulphite resistance to wine isolates. Surprisingly, this resulted in decreased sulphite resistance in the reference strain showing that the sole translocation is not the driver of increased resistance. Secondly, we found that shuffled strains had severely impaired spore viability and showed large phenotypic diversity in various stressful conditions leading in some instances to a strong fitness advantage, although no coding region was altered by the rearrangements. Therefore our method allows exploring the genotypic space accessible by structural variations and their phenotypic impact independently from the background effect.
Publisher
Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
