Genetic screens in isogenic mammalian cell lines without single cell cloning

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

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Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

10.1101/2020.05.18.102764 ◽

2020 ◽

Cited By ~ 5

Author(s):

Merve Dede ◽

Megan McLaughlin ◽

Eiru Kim ◽

Traver Hart

Keyword(s):

Cell Lines ◽

Protein Complexes ◽

Essential Genes ◽

Systematic Analysis ◽

Synthetic Lethal ◽

Genome Wide ◽

Synthetic Lethal Interactions ◽

Crispr Screen ◽

A Cell ◽

Genetic Buffering

AbstractMajor efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.

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Combinatorial CRISPR screen identifies fitness effects of gene paralogues

Nature Communications ◽

10.1038/s41467-021-21478-9 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Nicola A. Thompson ◽

Marco Ranzani ◽

Louise van der Weyden ◽

Vivek Iyer ◽

Victoria Offord ◽

...

Keyword(s):

Cancer Cells ◽

Cell Lines ◽

Unknown Function ◽

Single Copy ◽

Genetic Redundancy ◽

Synthetic Lethal ◽

Fitness Effects ◽

Synthetic Lethal Interactions ◽

Crispr Screen ◽

Multiple Cell

AbstractGenetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.

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Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Nucleic Acids Research ◽

10.1093/nar/gku576 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9838-9853 ◽

Cited By ~ 6

Author(s):

Saeed Kaboli ◽

Takuya Yamakawa ◽

Keisuke Sunada ◽

Tao Takagaki ◽

Yu Sasano ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Interaction ◽

Interaction Network ◽

Essential Genes ◽

Genetic Interactions ◽

Lethal Gene ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

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Cytotoxicity Study of Textile Fabrics Impregnated With CuO Nanoparticles in Mammalian Cells

International Journal of Toxicology ◽

10.1177/1091581817736712 ◽

2017 ◽

Vol 36 (6) ◽

pp. 478-484 ◽

Cited By ~ 9

Author(s):

Gagandeep Singh ◽

James Beddow ◽

Christopher Mee ◽

Lidia Maryniak ◽

Eadaoin M. Joyce ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cells ◽

Dermal Fibroblast ◽

Copper Oxide Nanoparticles ◽

Growth Media ◽

Indirect Contact ◽

Cuo Nps ◽

Mammalian Cell Lines ◽

Textile Fabrics ◽

Hdf Cells

Copper and copper compounds have multifunctional properties (antibacterial, antiviral, and antifungal) with promising applications. Copper in its nanoparticle (Cu NPs) forms has been widely used in various industrial and commercial applications. In the current research, the cytotoxic effects of textile fabrics impregnated with copper oxide nanoparticles (CuO NPs) were studied in mammalian cell lines. CuO NPs were impregnated onto textile substrates using 2 different techniques: the sonochemical generation and impregnation of NPs from metal complexes ( insitu) and a “throwing the stones” technology using commercially prepared CuO NPs. The cytotoxicity of these 2 textile fabric types was assayed on human dermal fibroblast (HDF) cells and human hepatocellular carcinoma cells (HepG2) and was evaluated by indirect contact using an MTT assay. The impregnated fabrics were not exposed to the cells, rather their leachates were used to test cytotoxicity. The fabrics were soaked into the growth media for up to 7 days, and the leachates from day 1 and day 7 were incubated with the cell lines for 24 hours prior to the testing. The discharge or leaching from antimicrobial nanomaterials into the surroundings and surface waters is posing a serious environmental threat, which needs to be addressed. Hence, with regard to product safety, it is a good approach to study the fabric leachates rather than the intact material. The results showed that CuO NPs are not toxic to HDF cells. However, cytotoxicity was seen in HepG2 cells with cell viability decreasing by 20% to 25% for all the fabrics after 24 hours.

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ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

Infection and Immunity ◽

10.1128/iai.00651-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4600-4608 ◽

Cited By ~ 40

Author(s):

Karin Heine ◽

Sascha Pust ◽

Stefanie Enzenmüller ◽

Holger Barth

Keyword(s):

Cell Death ◽

Cell Lines ◽

Mammalian Cells ◽

Clostridium Botulinum ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Adp Ribosylation ◽

Mammalian Cell Lines ◽

Adp Ribosyltransferase ◽

C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

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Cell Line Classification Using Electric Cell-Substrate Impedance Sensing (ECIS)

The International Journal of Biostatistics ◽

10.1515/ijb-2018-0083 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 4

Author(s):

Megan L. Gelsinger ◽

Laura L. Tupper ◽

David S. Matteson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Mammalian Cells ◽

Statistical Tests ◽

Classification Methods ◽

Impedance Sensing ◽

Mammalian Cell Lines ◽

Cell Substrate ◽

Multiple Cell ◽

Line Classification

AbstractWe present new methods for cell line classification using multivariate time series bioimpedance data obtained from electric cell-substrate impedance sensing (ECIS) technology. The ECIS technology, which monitors the attachment and spreading of mammalian cells in real time through the collection of electrical impedance data, has historically been used to study one cell line at a time. However, we show that if applied to data from multiple cell lines, ECIS can be used to classify unknown or potentially mislabeled cells, factors which have previously been associated with the reproducibility crisis in the biological literature. We assess a range of approaches to this new problem, testing different classification methods and deriving a dictionary of 29 features to characterize ECIS data. Most notably, our analysis enriches the current field by making use of simultaneous multi-frequency ECIS data, where previous studies have focused on only one frequency; using classification methods to distinguish multiple cell lines, rather than simple statistical tests that compare only two cell lines; and assessing a range of features derived from ECIS data based on their classification performance. In classification tests on fifteen mammalian cell lines, we obtain very high out-of-sample predictive accuracy. These preliminary findings provide a baseline for future large-scale studies in this field.

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Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

Genome Biology ◽

10.1186/s13059-020-02173-2 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 2

Author(s):

Merve Dede ◽

Megan McLaughlin ◽

Eiru Kim ◽

Traver Hart

Keyword(s):

Cell Lines ◽

Protein Complexes ◽

Gene Knockout ◽

Essential Genes ◽

Loss Of Function ◽

Systematic Analysis ◽

Synthetic Lethal ◽

Synthetic Lethal Interactions ◽

Crispr Screen ◽

A Cell

Abstract Background Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. Results Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Conclusions Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.

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Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity

Canadian Journal of Microbiology ◽

10.1139/m77-026 ◽

1977 ◽

Vol 23 (2) ◽

pp. 183-189 ◽

Cited By ~ 78

Author(s):

John L. Middlebrook ◽

Rebecca B. Dorland

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Lines ◽

Mammalian Cells ◽

Corynebacterium Diphtheriae ◽

Pseudomonas Exotoxin ◽

Intact Cells ◽

Mammalian Cell Lines ◽

First Order Kinetics ◽

Wide Range ◽

Species Specific

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1–4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat-and mouse-derived cell lines were very in sensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line–specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.

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Reticulon 4a promotes exocytosis in mammalian cells

Molecular Biology of the Cell ◽

10.1091/mbc.e19-03-0159 ◽

2019 ◽

Vol 30 (18) ◽

pp. 2349-2357 ◽

Cited By ~ 2

Author(s):

Richik Nilay Mukherjee ◽

Daniel L. Levy

Keyword(s):

Cell Surface ◽

Protein Trafficking ◽

Mammalian Cells ◽

Cell Types ◽

Vesicular Trafficking ◽

Secreted Proteins ◽

Mammalian Cell Lines ◽

Rough Er ◽

Different Cell Types ◽

Functional Output

Endoplasmic reticulum (ER) tubules and sheets conventionally correspond to smooth and rough ER, respectively. The ratio of ER tubules-to-sheets varies in different cell types and changes in response to cellular conditions, potentially impacting the functional output of the ER. To directly test whether ER morphology impacts vesicular trafficking, we increased the tubule-to-sheet ratio in three different ways, by overexpressing Rtn4a, Rtn4b, or REEP5. Only Rtn4a overexpression increased exocytosis, but not overall levels, of several cell surface and secreted proteins. Furthermore, Rtn4a depletion reduced cell surface trafficking without affecting ER morphology. Similar results were observed in three different mammalian cell lines, suggesting that Rtn4a generally enhances exocytosis independently of changes in ER morphology. Finally, we show that Rtn4a levels modulate cell adhesion, possibly by regulating trafficking of integrins to the cell surface. Taking the results together, we find that altering ER morphology does not necessarily affect protein trafficking, but that Rtn4a specifically enhances exocytosis.

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