Faculty Opinions recommendation of Gene essentiality and synthetic lethality in haploid human cells.

SUMMARYForward genetic screens across hundreds of diverse cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly resemble metabolite availability in human blood. To explore how medium composition influences gene essentiality, we performed CRISPR-based screens of human cancer cell lines cultured in traditional versus human plasma-like medium (HPLM). Sets of medium-dependent fitness genes span several cellular processes and can vary with both natural cell-intrinsic diversity and the specific combination of basal and serum components that comprise typical culture media. Notably, we traced the causes for each of three conditional growth phenotypes to the availability of metabolites uniquely defined in HPLM versus traditional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.

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Genetic synthetic lethality screen at the single gene level in cultured human cells

Nucleic Acids Research ◽

10.1093/nar/29.20.e100 ◽

2001 ◽

Vol 29 (20) ◽

pp. 100e-100 ◽

Cited By ~ 14

Author(s):

A. H. Simons

Keyword(s):

Synthetic Lethality ◽

Single Gene ◽

Human Cells ◽

Gene Level ◽

Cultured Human Cells

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Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

10.1101/2020.04.25.061135 ◽

2020 ◽

Author(s):

Luisa Henkel ◽

Benedikt Rauscher ◽

Barbara Schmitt ◽

Jan Winter ◽

Michael Boutros

Keyword(s):

Experimental Design ◽

Single Cell ◽

Dynamic Range ◽

High Sensitivity ◽

Human Cells ◽

Gene Essentiality ◽

Cell Clones ◽

Experimental Implementation ◽

Genome Scale ◽

Target Activity

ABSTRACTGiven their broad utility in functionally annotating genomes, the experimental design of genome-scale CRISPR screens can vary greatly and criteria for optimal experimental implementation and library composition are still emerging. In this study, we report advantages of conducting viability screens in selected Cas9 single cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg)RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a new genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. In summary, we show how empirically designed libraries in combination with an optimised experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

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Establishment of a Chemical Synthetic Lethality Screen in Cultured Human Cells

Genome Research ◽

10.1101/gr.154201 ◽

2001 ◽

Vol 11 (2) ◽

pp. 266-273 ◽

Cited By ~ 31

Author(s):

A. Simons

Keyword(s):

Synthetic Lethality ◽

Human Cells ◽

Cultured Human Cells

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Alpha-Tocopherol Protects Cultured Human Cells from the Acute Lethal Cytotoxicity of Dioxin

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831.72.3.147 ◽

2002 ◽

Vol 72 (3) ◽

pp. 147-153 ◽

Cited By ~ 8

Author(s):

Kei-Ichi Hirai ◽

Jie-Hong Pan ◽

Ying-Bo Shui ◽

Eriko Simamura ◽

Hiroki Shimada ◽

...

Keyword(s):

Cell Death ◽

Cell Damage ◽

Smooth Endoplasmic Reticulum ◽

Dehydroascorbic Acid ◽

Human Cells ◽

Alpha Tocopherol ◽

Cervical Cancer Cells ◽

Cultured Human Cells ◽

Nuclear Damage ◽

Conjunctival Epithelial Cells

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30–40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0–31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78–97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

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