Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements.
Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements.
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使用针对转座TY1元素的CRISPR/CAS9生成的DSB将酵母基因组改组。
DOI：
10.1371/journal.pgen.1010590
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发表时间：
2023-01
期刊：
影响因子：
4.5
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中科院分区：
文献类型：
--
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尽管转座元件之间的同源重组可以通过促进染色体重排来驱动酵母中的基因组进化，但潜在机制的细节尚未完全阐明。在酿酒酵母的基因组中，最常见的一类转座子是逆转录转座子 Ty1。在这里，我们探讨了 Cas9 诱导的针对 Ty1 元件的双链断裂 (DSB) 如何在该酵母物种中产生基因组改变。 Cas9 诱导后，我们观察到染色体重排（例如缺失、重复和易位）显着增加。此外，我们发现有丝分裂重组率升高，导致杂合性丧失。使用 Southern 分析结合短读长和长读长 DNA 测序，我们揭示了逆转录转座子诱导重组的重要特征。几乎所有的染色体重排都反映了非等位同源重组对 Ty1 元件 DSB 的修复；聚集的 Ty 元件是染色体重排的热点。相反，很大一部分（约四分之三）等位基因有丝分裂重组事件在独特序列中具有断点。我们的分析表明，后面的一些事件反映了对 Ty 元件中产生的断裂末端的广泛加工，这些断裂末端延伸到独特的序列中，从而导致断裂诱导的复制。最后，我们发现单倍体和二倍体菌株对于修复双链DNA断裂的途径有不同的偏好。我们的研究结果证明了逆转录转座子中 DNA 损伤在驱动基因组进化中的重要性。转座因子可以影响基因表达和基因功能。位于同一染色体上的元件之间的重组可以产生缺失、重复和倒位，而非同源染色体上的转座元件之间的重组可以产生易位。因此，转座因子之间的重组是遗传变异的重要原因。在本报告中，我们对通过 CRISPR/Cas9 靶向反转录转座子 Ty1 家族在酿酒酵母单倍体和二倍体菌株中诱导的重组事件进行了全面分析。染色体重排被刺激超过 1000 倍。使用传统短“读段”和超长“读段”的 DNA 测序表明，几乎所有这些重排的断点处都有 Ty1 元件。相比之下，我们还观察到同源物之间等位基因有丝分裂重组事件的刺激，其中大多数不涉及直接的 Ty-Ty 相互作用。我们的结果为重复序列调控的基因组进化机制提供了新的见解。
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. Transposable elements can affect gene expression and gene function. Recombination between elements located on the same chromosome can produce deletions, duplications, and inversions, whereas recombination between transposable elements on non-homologous chromosomes can generate translocations. Thus, recombination between transposable elements is an important cause of genetic variation. In this report, we present a comprehensive analysis of recombination events induced in both haploid and diploid strains of Saccharomyces cerevisiae by targeting the Ty1 family of retrotransposons with CRISPR/Cas9. Chromosome rearrangements were stimulated more than 1000-fold. DNA sequencing, using both conventional short “reads” and ultra-long “reads”, showed that almost all of these rearrangements had Ty1 elements at their breakpoints. In contrast, we also observed a stimulation of allelic mitotic recombination events between homologs, most of which did not involve direct Ty-Ty interactions. Our results provided novel insights into the mechanism of genome evolution as regulated by repetitive sequences.
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影响因子：
7
作者：
McGinty RJ;Rubinstein RG;Neil AJ;Dominska M;Kiktev D;Petes TD;Mirkin SM
通讯作者：
Mirkin SM
影响因子：
48
作者：
Chin, Chen-Shan;Peluso, Paul;Schatz, Michael C.
通讯作者：
Schatz, Michael C.
影响因子：
7
作者：
Kim, JM;Vanguri, S;Voytas, DF
通讯作者：
Voytas, DF
DOI：
10.1126/science.1191125
发表时间：
2010-07-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hicks WM;Kim M;Haber JE
通讯作者：
Haber JE
影响因子：
64.5
作者：
ERREDE, B;CARDILLO, TS;WIAME, JM
通讯作者：
WIAME, JM