Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A.; Nelson, Angelique M.; Hookway, Caroline; Ludmann, Susan A.; Mueller, Irina A.; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M.; Gunawardane, Ruwanthi N.

doi:10.1091/mbc.e17-03-0209

Cited by 226 publications

(280 citation statements)

References 72 publications

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“…In order to test this, cells were passaged for >30 passages and protein expression levels compared to the earlier passage number (Supporting Information Figure 1c). This suggests that the cells express a lower level of TPX2-EGFP than the untagged TPX2, consistent with observations of other CRISPR tagged genes (Roberts et al, 2017). This suggests that the cells express a lower level of TPX2-EGFP than the untagged TPX2, consistent with observations of other CRISPR tagged genes (Roberts et al, 2017).…”

Section: Generating Crispr Cells For Protein Localizationsupporting

confidence: 88%

“…Several groups have demonstrated the feasibility of this method, showing that the tagged proteins localize to the expected structures in cells without overexpression artifacts (Cho et al, 2016;Dean & Palmer, 2014;Gan et al, 2016;Hsu, Lander, & Zhang, 2014;Ratz, Testa, Hell, & Jakobs, 2015;Roberts et al, 2017). Several groups have demonstrated the feasibility of this method, showing that the tagged proteins localize to the expected structures in cells without overexpression artifacts (Cho et al, 2016;Dean & Palmer, 2014;Gan et al, 2016;Hsu, Lander, & Zhang, 2014;Ratz, Testa, Hell, & Jakobs, 2015;Roberts et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

Cytoskeleton

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The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

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Section: Generating Crispr Cells For Protein Localizationsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

Cytoskeleton

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“…While optimizing transient transfection using mito-YFP is helpful at reducing unwanted side-effects, creating a stable cell line using lentiviral or retroviral methods, although time consuming, is the best option to reduce the possibility of the mitochondrial fluorescent protein interfering with mitochondrial morphology [38]. Another method to ensure low expression uses the CRISPR/Cas9 technology to insert fluorescent protein tags into endogenous loci [87]. The Allen Institute deposited a variety of these constructs at Addgene, including the mitochondrial protein Tom20 tagged with mEGFP (Table 3).…”

Section: 3 Resultsmentioning

confidence: 99%

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

Harwig

Viana

Egner

et al. 2018

Analytical Biochemistry

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Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae. Here Mitograph was assessed on five different mammalian cells types, all of which were accurately segmented by MitoGraph analysis. MitoGraph also successfully differentiated between distinct mitochondrial morphologies that ranged from entirely fragmented to hyper-elongated. General recommendations are also provided for confocal imaging of labeled mitochondria (using mito-YFP, MitoTracker dyes and immunostaining parameters). Widespread adoption of MitoGraph will help achieve a long-sought goal of consistent and reproducible quantification of mitochondrial morphology.

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“…This approach will provide matched isogenic controls for HTI screening purposes, thus isolating the contribution of specific pathogenic mutations from confounding effects due to heterogeneity in genetic backgrounds of iPS cell donors. Furthermore, CRISPR/Cas9 will also be instrumental to label endogenous proteins with genetically encoded fluorescent tags in iPS cells [73], potentially on a large scale [74,75], with the goal of generating novel HTI-based assays to be used in chemical or RNAi screens.…”

Section: Concluding Remarks: Challenges and Future Directionsmentioning

confidence: 99%

High-Throughput Imaging for the Discovery of Cellular Mechanisms of Disease

Pegoraro

Misteli

2017

Trends in Genetics

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High-throughput imaging (HTI) is a promising tool in the discovery of cellular disease mechanisms. While traditional approaches to identify disease pathways often rely on knowledge of the causative genetic defect, HTI-based screens offer an unbiased discovery approach based on any morphological or functional defects of disease cells or tissues. Here we provide an overview of the use of HTI for the study of human disease mechanisms. We discuss key technical aspects of HTI and highlight representative examples of its practical applications for the discovery of molecular mechanisms of disease, focusing on infectious diseases and host-pathogen interactions, cancer, and a rare genetic disease. We also present some of the current challenges and possible solutions offered by recent novel cell culture systems and genome engineering approaches.

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Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

Abstract: The generation of a collection of human induced pluripotent stem cell (hiPSC) lines expressing endogenously GFP-tagged proteins using CRISPR/Cas9 methods is described. The methods used and the genomic and cell biological data validating the GFP-tagged hiPSC lines are also presented.

Cited by 226 publications

References 72 publications

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

High-Throughput Imaging for the Discovery of Cellular Mechanisms of Disease

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