Synthetic Lethality Screens Using RNAi in Combination with  CRISPR-based Knockout in Drosophila Cells

Housden, Benjamin E.; Nicholson, Hilary E.; Perrimon, Norbert

doi:10.21769/bioprotoc.2119

Cited by 13 publications

(8 citation statements)

References 6 publications

(6 reference statements)

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“…In fact, research of the past decade has made it abundantly clear that complex genetic interactions (i.e. buffering relationships between genes) play a crucial role in modulating the genotype-phenotype relationship ( Baryshnikova et al, 2013 ; Butland et al, 2008 ; Byrne et al, 2007 ; Costanzo et al, 2016 ; Housden et al, 2017 ). Fortunately, methodologies capable of characterizing genetic interactions at the genome-wide level have been developed to aid researchers in grappling with this complexity ( Dixon et al, 2009b ; Kuzmin et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

“…In this way, functional relationships between the query gene and all other genes in the genome can be characterized in a systematic and unbiased manner. Such strategies have been used successfully in a wide variety of model systems including Saccharomyces cerevisiae , S. pombe , Escherichia coli , Caenorhabditis elegans , Drosophila melanogaster , and more recently in mammalian cell lines ( Butland et al, 2008 ; Byrne et al, 2007 ; Costanzo et al, 2016 ; Housden et al, 2015 , 2017 ; Ryan et al, 2012 ; Shen et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function

et al. 2017

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Tuberous sclerosis complex is an autosomal dominant disorder characterized by benign tumors arising from the abnormal activation of mTOR signaling in cells lacking TSC1 (hamartin) or TSC2 (tuberin) activity. To expand the genetic framework surrounding this group of growth regulators, we utilized the model eukaryote Schizosaccharomyces pombe to uncover and characterize genes that buffer the phenotypic effects of mutations in the orthologous tsc1 or tsc2 loci. Our study identified two genes: fft3 (encoding a DNA helicase) and ypa1 (encoding a peptidyle-prolyl cis/trans isomerase). While the deletion of fft3 or ypa1 has little effect in wild-type fission yeast cells, their loss in tsc1Δ or tsc2Δ backgrounds results in severe growth inhibition. These data suggest that the inhibition of Ypa1p or Fft3p might represent an ‘Achilles’ heel’ of cells defective in hamartin/tuberin function. Furthermore, we demonstrate that the interaction between tsc1/tsc2 and ypa1 can be rescued through treatment with the mTOR inhibitor, torin-1, and that ypa1Δ cells are resistant to the glycolytic inhibitor, 2-deoxyglucose. This identifies ypa1 as a novel upstream regulator of mTOR and suggests that the effects of ypa1 loss, together with mTOR activation, combine to result in a cellular maladaptation in energy metabolism that is profoundly inhibitory to growth.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function

et al. 2017

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“…We store conditioned medium at 4°C and use it for as long as 1 month. Also see Housden, Nicholson, & Perrimon, 2017 Schneiders' medium with 2 µg/ml puromycin Dilute a 10 mg/ml puromycin (Calbiochem #540411) stock solution to a final concentration of 2 µg/ml (1:5000) in Schneider's medium (see recipe).…”

Section: Conditioned Mediummentioning

confidence: 99%

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

Bosch

Knight

Kanca

et al. 2019

CP Molecular Biology

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The CRISPR‐Cas9 system makes it possible to cause double‐strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or ‘knock‐in’ of an exogenous cassette. One common application of knock‐in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock‐in of fluorescent protein ORFs into Cas9‐expressing Drosophila S2R+ cultured cells, the single‐stranded DNA (ssDNA) Drop‐In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop‐In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock‐in into Cas9‐positive S2R+ cells using the ssDNA Drop‐In approach Basic Protocol 2: Knock‐in into Cas9‐positive S2R+ cells by homology‐independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+‐MT::Cas9 Drosophila cells Support Protocol 5: Single‐cell isolation of fluorescent cells using FACS

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“…Towards this goal, we reported previously the use of a cross-species screening approach to identify SS/L interactions with TSC1 and TSC2, the two tumor suppressor genes underlying tuberous sclerosis complex (TSC) (Housden et al 2015(Housden et al , 2017. We performed focused dsRNA screens targeting all kinases and phosphatases in wild-type, TSC1 and TSC2 mutant Drosophila cells.…”

Section: Introductionmentioning

confidence: 99%

Variable dose analysis: A novel RNAi-based method for detection of synthetic lethal interactions

Housden

Kelley

et al. 2017

Preprint

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Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells but such screens generally suffer from low reproducibility. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency.Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called variable dose analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC) and generated a SS/L network for TSC. Using this network, we identified four FDA-approved drugs that selectively affect viability of TSC deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.

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Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells

Cited by 13 publications

References 6 publications

Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function

Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

Variable dose analysis: A novel RNAi-based method for detection of synthetic lethal interactions

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