Optical Pooled Screens in Human Cells

Feldman, David; Singh, Avtar; Schmid-Burgk, Jonathan L.; Carlson, Robert F.; Mezger, Anja; Garrity, Anthony J.; Zhang, Feng; Blainey, Paul C.

doi:10.1016/j.cell.2019.09.016

Cited by 217 publications

(178 citation statements)

References 77 publications

(80 reference statements)

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“…S1; Sabari et al 2018). The application of the CROP-seq vector (Datlinger et al 2017) that expresses the sgRNA sequence in a polyadenylated mRNA transcript enabled us to cell-specifically identify the targeted intron by situ sequencing (Larsson et al 2010;Ke et al 2013;Feldman et al 2019). To identify the sgRNA sequence integrated into individual cells, we fixed the cell pool and developed a two-color in situ sequencing protocol compatible with the presence of the GFP tag ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time

2020

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The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

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Section: Resultsmentioning

confidence: 99%

Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time

2020

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“…Two other methods have recently been developed to use imaging both for phenotypic screening and decoding to permit sgRNA identification in individual cells in situ 15,16 . In both methods, CRISPR sgRNA expression constructs were modified to express both a sgRNA and a barcode.…”

Section: Optical Enrichment Compared To Other Methods For Phenotypicmentioning

confidence: 99%

“…The barcode can be read out either by in situ sequencing 15 20 . This method also can use most available sgRNA libraries and is compatible with further live cell studies.…”

Section: Optical Enrichment Compared To Other Methods For Phenotypicmentioning

confidence: 99%

“…However, many important cellular phenotypes can only be detected under a microscope, which requires a robust method for transforming optically identified phenotypes into differences in sgRNA abundance. Recently, several in situ sequencing 15,16 and cell isolation methods [17][18][19][20] were developed which allow microscopes to be used for screening. However, these methods contain non-high throughput steps that limit their scalability.…”

Section: Introductionmentioning

confidence: 99%

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High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

Yan

Stuurman

Ribeiro

et al. 2020

Preprint

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ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeats) -based gene inactivation provides a powerful means of linking genes to particular cellular phenotypes. CRISPR-based screening has mostly relied upon using large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to computationally select cells displaying a particular CRISPR-induced phenotype, mark them by photo-conversion of an expressed photo-activatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photo-conversion of cells, allowing ~1.5 million individual cells to be screened in 8 hr. We used this approach to screen 6092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a highly scalable approach to facilitate imaging-based pooled CRISPR screens.

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“…Fluorescent activated cell sorting (FACS) or high-throughput microscopy can then be utilized to measure the response of the cells to the genetic or chemical perturbations. Until recently, microscopic readouts were restricted to arrayed screens, but new technologies have adapted this approach to make it amenable to pooled screens as well [215]. To date, multiple optical readouts have been established to study DNA damage and repair.…”

Section: Outlook: Approaches To Identify Interactions Between Metabolmentioning

confidence: 99%

Interplay between Cellular Metabolism and the DNA Damage Response in Cancer

Moretton

Loizou

2020

Cancers

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Metabolism is a fundamental cellular process that can become harmful for cells by leading to DNA damage, for instance by an increase in oxidative stress or through the generation of toxic byproducts. To deal with such insults, cells have evolved sophisticated DNA damage response (DDR) pathways that allow for the maintenance of genome integrity. Recent years have seen remarkable progress in our understanding of the diverse DDR mechanisms, and, through such work, it has emerged that cellular metabolic regulation not only generates DNA damage but also impacts on DNA repair. Cancer cells show an alteration of the DDR coupled with modifications in cellular metabolism, further emphasizing links between these two fundamental processes. Taken together, these compelling findings indicate that metabolic enzymes and metabolites represent a key group of factors within the DDR. Here, we will compile the current knowledge on the dynamic interplay between metabolic factors and the DDR, with a specific focus on cancer. We will also discuss how recently developed high-throughput technologies allow for the identification of novel crosstalk between the DDR and metabolism, which is of crucial importance to better design efficient cancer treatments.

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Optical Pooled Screens in Human Cells

Abstract: Highlights d In situ sequencing of perturbations or barcodes enables image-based pooled screens d p65 translocation is assayed by imaging in fixed and live cell pools d Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-kB

Cited by 217 publications

References 77 publications

Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time

Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time

High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

Interplay between Cellular Metabolism and the DNA Damage Response in Cancer

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