Related Experiment Video
Updated: Aug 12, 2025
09:29
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
1.2K
Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements.
Lei Qi1,2, Yang Sui1,2, Xing-Xing Tang1
1Ocean College, Zhejiang University, Zhoushan, China.
Plos Genetics
|January 26, 2023
Summary
DNA double-strand breaks (DSBs) in yeast retrotransposons (Ty1 elements) drive significant genomic evolution. Repair mechanisms, including non-allelic homologous recombination and break-induced replication, lead to chromosome rearrangements and loss of heterozygosity.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Genomics
Background:
- Homologous recombination involving transposable elements is a known driver of genomic evolution in yeast.
- The retrotransposon Ty1 is the most prevalent transposon in the Saccharomyces cerevisiae genome.
- Mechanisms by which transposable elements induce genomic alterations remain incompletely understood.
Purpose of the Study:
- To investigate the genomic alterations resulting from Cas9-induced double-strand breaks (DSBs) targeted to Ty1 elements in Saccharomyces cerevisiae.
- To elucidate the repair pathways involved in processing DSBs within Ty1 elements and their impact on genome stability.
- To compare DNA repair pathway preferences in haploid and diploid yeast strains.
Main Methods:
- Induction of Cas9-mediated DSBs specifically at Ty1 elements in Saccharomyces cerevisiae.
- Analysis of chromosomal rearrangements (deletions, duplications, translocations) and mitotic recombination using Southern analysis.
- High-throughput DNA sequencing (short- and long-read) to identify breakpoints and characterize recombination events.
- Comparative analysis of repair pathway usage in haploid versus diploid yeast strains.
Main Results:
- Cas9 induction at Ty1 elements significantly elevated chromosomal rearrangements and mitotic recombination, leading to loss of heterozygosity.
- Chromosomal rearrangements primarily resulted from non-allelic homologous recombination repair of DSBs at Ty1 elements, with clustered Ty elements acting as hotspots.
- A substantial proportion of allelic mitotic recombination events involved breakpoints in unique sequences, suggesting extensive end processing and break-induced replication.
- Haploid and diploid yeast strains exhibited distinct preferences for DNA double-strand break repair pathways.
Conclusions:
- DNA lesions within retrotransposons, specifically Ty1 elements, are critical mediators of genome evolution in yeast.
- Non-allelic homologous recombination and break-induced replication are key mechanisms by which Ty1-associated DSBs induce genomic alterations.
- Strain ploidy influences the choice of DNA double-strand break repair pathways, highlighting the complexity of genome maintenance.
Related Concept Videos
01:28CRISPR/Cas9 Genome Editing
113
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
113
02:53Conservative Site-specific Recombination and Phase Variation
6.1K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.1K
01:59CRISPR
52.6K
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
52.6K