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Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

ABSTRACT: We explored how Cas9-induced double-strand breaks (DSBs) on Ty1 produce genomic alterations in the diploid yeast Saccharomyces cerevisiae. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements (large deletions and duplications), loss of heterozygosity (gene conversions, crossovers, and break-induced replication), and aneuploidy. Almost all of the chromosomal rearrangements reflect the repairing of DSBs at Ty1 elements by homologous recombination.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE205199 | GEO | 2022/06/01

REPOSITORIES: GEO

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Project description:The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. In addition to examining crossovers on chromosome V, we mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

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Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

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