Identification of genes that are essential to restrict genome duplication to once per cell division

Vassilev, Alex; Lee, Chrissie Y.; Vassilev, Boris; Zhu, Wenge; Ormanoglu, Pinar; Martin, Scott E.; DePamphilis, Melvin L.

doi:10.18632/oncotarget.9008

“…We first obtained HCT-116 cells stably expressing Cas9 protein to evaluate synthetic CRISPR, as the knockdown of a several control genes, including GMNN , results in enlarged nuclei with increased DNA content in HCT-116 cells[23, 24]. HCT-116 Cas9 cells were then reverse transfected with crRNA:tracrRNA complexes targeting GMNN and monitored for changes in nuclear size.…”

Section: Resultsmentioning

confidence: 99%

“…We evaluated additional crRNAs targeting several genes known to yield enlarged nuclei and increased DNA stain intensity upon knockdown in HCT-116 cells[24]. Five crRNAs were tested per gene.…”

Section: Resultsmentioning

confidence: 99%

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Tan¹,

Martin²

2016

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To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.

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“…Reentry into S-phase can occur multiple times and can lead to ploidy shifts from 1N to 32N (71). Interestingly, 42 uncharacterized genes were recently identified in human cells for their role in preventing endoreplication (72), supporting that DNA replication controls are still being discovered.…”

Section: Mutations Underlying Ploidy Amplificationmentioning

confidence: 95%

Ploidy Variation in Fungi: Polyploidy, Aneuploidy, and Genome Evolution

Todd

¹

,

Forche

²

,

Selmecki

³

2017

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The ability of an organism to replicate and segregate its genome with high fidelity is vital to its survival and for the production of future generations. Errors in either of these steps (replication or segregation) can lead to a change in ploidy or chromosome number. While these drastic genome changes can be detrimental to the organism, resulting in decreased fitness, they can also provide increased fitness during periods of stress. A change in ploidy or chromosome number can fundamentally change how a cell senses and responds to its environment. Here, we discuss current ideas within fungal biology that illuminate how eukaryotic genome size variation can impact the organism at a cellular and evolutionary level. One of the most fascinating observations from the last two decades of research is that some fungi have evolved the ability to tolerate large genome size changes and generate vast genomic heterogeneity without undergoing canonical meiosis.

show abstract

“…Reentry into S-phase can occur multiple times and can lead to ploidy shifts from 1N to 32N (71). Interestingly, 42 uncharacterized genes were recently identified in human cells for their role in preventing endoreplication (72), supporting that DNA replication controls are still being discovered.…”

Section: Ploidy Changes In the Context Of Laboratory Mutants: Towamentioning

confidence: 97%

Ploidy Variation in Fungi: Polyploidy, Aneuploidy, and Genome Evolution

Todd

¹

,

Forche

²

,

Selmecki

³

2017

View full text Add to dashboard Cite

The ability of an organism to replicate and segregate its genome with high fidelity is vital to its survival and for the production of future generations. Errors in either of these steps (replication or segregation) can lead to a change in ploidy or chromosome number. While these drastic genome changes can be detrimental to the organism, resulting in decreased fitness, they can also provide increased fitness during periods of stress. A change in ploidy or chromosome number can fundamentally change how a cell senses and responds to its environment. Here, we discuss current ideas within fungal biology that illuminate how eukaryotic genome size variation can impact the organism at a cellular and evolutionary level. One of the most fascinating observations from the last two decades of research is that some fungi have evolved the ability to tolerate large genome size changes and generate vast genomic heterogeneity without undergoing canonical meiosis.

show abstract

Identification of genes that are essential to restrict genome duplication to once per cell division

Cited by 26 publications

References 81 publications

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Ploidy Variation in Fungi: Polyploidy, Aneuploidy, and Genome Evolution

Ploidy Variation in Fungi: Polyploidy, Aneuploidy, and Genome Evolution

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