Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes

Ihry, Robert J.; Salick, Max R.; Ho, Daniel J.; Sondey, Marie; Kommineni, Sravya; Paula, Steven; Raymond, Joe; Henry, Beata; Frias, Elizabeth; Wang, Qiong; Worringer, Kathleen A.; Ye, Chaoyang; Russ, Carsten; Reece‐Hoyes, John S.; Altshuler, Robert C.; Randhawa, Ranjit; Yang, Zinger; McAllister, Gregory; Hoffman, Gregory R.; Dolmetsch, Ricardo; Kaykas, Ajamete

doi:10.1016/j.celrep.2019.03.043

Cited by 67 publications

(72 citation statements)

References 73 publications

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“…Critically, the DepMap screens were performed using the Avana gRNA library (Doench et al, 2016), indicating that our findings are not specific to our own CRISPR libraries. Similarly, Ihry et al (2019) reported successful genomescale CRISPR screens in a TP53 wild-type human pluripotent stem cell line, despite observing considerable p53-mediated Cas9 toxicity in these cells (Ihry et al, 2018), which is in agreement with data from our own laboratory in a comparable system (Mair et al, 2019). In summary, while the induction of a p53 response in TP53 wildtype cells is not unexpected, it does not hamper the screenability of TP53 wild-type cells in general.…”

supporting

confidence: 84%

CRISPR screens are feasible in TP 53 wild‐type cells

Brown

Mair

Soste

et al. 2019

Molecular Systems Biology

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A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR ‐based functional genomic screens. Brown et al report good performance of genome‐scale screens in TP 53 wild‐type cells and reiterate best practices for CRISPR screening.

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supporting

confidence: 84%

CRISPR screens are feasible in TP 53 wild‐type cells

Brown

Mair

Soste

et al. 2019

Molecular Systems Biology

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“…One positively selected set is related to the p53 pathway ("TP53 Network"), which is expected, as it is known that disabling p53-mediated DNA repair signaling increases proliferation in iPSCs (Haapaniemi et al 2018;Ihry et al 2018) . Several of the genes positively selected in iPSCs upon perturbation (PMAIP1, TP53, CHECK2) were recently individually confirmed in independent studies of stem cell specific essential genes Ihry et al 2019) .…”

Section: Double Grna Library Enables Screens In Ipscsmentioning

confidence: 88%

Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

Peets

Crepaldi

Zhou

et al. 2019

Preprint

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these authors contributed equally to this work Genetic screens based on CRISPR/Cas technology are a powerful tool for understanding cellular phenotypes. However, the coverage and replicate requirements result in large experiment sizes, which are limiting when samples are scarce, or the protocols are expensive and laborious. Here, we present an approach to reduce the scale of genome-wide perturbation screens up to fivefold without sacrificing performance. To do so, we deliver two randomly paired gRNAs into each cell, and rely on recent advances in gRNA design, as well as availability of gRNA effect measurements, to reduce the number of gRNAs per gene. We designed a human genome-wide library that has effective size of 30,000 constructs, yet targets each gene with three gRNAs. Our minimized double guide RNA library gives similar results to a standard single gRNA one, but using substantially fewer cells. We demonstrate that genome-wide screens can be optimized in a demanding model of induced pluripotent stem cells, reducing reagent cost 70% per replicate compared to conventional approach, while retaining high performance. The screen design and the reduction in scale it provides will enable functional genomics experiments across many possible combinations of environments and genetic backgrounds, as well as in hard to obtain and culture primary cells.

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“…Our method may offer a route to find the best gene modulations (overexpression or partial knockdown) to carry out on multiple genes and towards multiple cellular objectives. For instance, expression vectors found with our method can be potentially used as a guide for CRISPR-Cas or CombiGEM systems [66], which have already proven successful in editing and modulating expression simultaneously across the genome [67], [68], e.g. to prioritize therapeutic targets in cancer cells [69].…”

Section: Discussionmentioning

confidence: 99%

Discovering Essential Multiple Gene Effects Through Large Scale Optimization: An Application to Human Cancer Metabolism

Occhipinti

Hamadi²,

Kugler

et al. 2021

IEEE/ACM Trans. Comput. Biol. and Bioinf.

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Computational modelling of metabolic processes has proven to be a useful approach to formulate our knowledge and improve our understanding of core biochemical systems that are crucial to maintaining cellular functions. Towards understanding the broader role of metabolism on cellular decision-making in health and disease conditions, it is important to integrate the study of metabolism with other core regulatory systems and omics within the cell, including gene expression patterns.After quantitatively integrating gene expression profiles with a genome-scale reconstruction of human metabolism, we propose a set of combinatorial methods to reverse engineer gene expression profiles and to find pairs and higher-order combinations of genetic modifications that simultaneously optimize multiobjective cellular goals. This enables us to suggest classes of transcriptomic profiles that are most suitable to achieve given metabolic phenotypes.We demonstrate how our techniques are able to compute beneficial, neutral or "toxic" combinations of gene expression levels. We test our methods on nine tissue-specific cancer models, comparing our outcomes with the corresponding normal cells, identifying genes as targets for potential therapies. Our methods open the way to a broad class of applications that require an understanding of the interplay among genotype, metabolism, and cellular behaviour, at scale.

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Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes

Cited by 67 publications

References 73 publications

CRISPR screens are feasible in TP 53 wild‐type cells

CRISPR screens are feasible in TP 53 wild‐type cells

Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

Discovering Essential Multiple Gene Effects Through Large Scale Optimization: An Application to Human Cancer Metabolism

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