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

Abstract

Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

“…For instance, a break outside a Ty element is more prone to generating rearrangements than a DSB induced within ( Hoang et al 2010 ). Also, the recent observation that a large proportion of recombination events induced by DSBs within LTR or Ty elements has breakpoints in unique sequences and generates frequent recombination between nontargeted repeats ( Fleiss et al 2019 ; Qi et al 2023 ) is consistent with the induction and repair of secondary DSBs, as shown for MIR ( Fig. 7 B–D).…”

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

“…For instance, a break outside a Ty element is more prone to generate rearrangements than a DSB induced within (Hoang et al 2010). Also, the recent observation that a large proportion of recombination events induced by DSBs within LTR or Ty elements have breakpoints in unique sequences and generate frequent recombination between non-targeted repeats (Qi et al 2023;Fleiss et al 2019) is consistent with the induction of secondary DSBs, as shown for MIR.…”

Nonlethal Furfural Exposure Causes Genomic Alterations and Adaptability Evolution in Saccharomyces cerevisiae

“…4A , the SNP microarray pattern showed that two chromosomal regions (bp 1 to 635650 of chromosome II and bp 1 to 182683 of chromosome XIII) were amplified, and two regions (bp 644285 to the right end of chromosome II and bp 183803 to the right end of chromosome XIII) were deleted in isolate LF4. According to our previous work ( 26 ), the majority of “paired” terminal deletions and duplications are the results of translocations between a centromere-containing fragment and an acentric fragment to form monocentric recombined chromosomes. In the LF4 lane of the pulsed-field gel electrophoresis (PFGE) results, a new band with a size of 818 kb was detected ( Fig.…”