Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior

Naxerova, Kamila; Stefano, Bruno Di; Makofske, Jessica; Watson, Emma V.; Kort, Marit AC de; Martin, Timothy D.; Dezfulian, Mohammed; Ricken, Dominik; Wooten, Eric C.; Kuroda, Mitzi I.; Hochedlinger, Konrad; Elledge, Stephen J.

doi:10.1101/gad.349048.121

Cited by 18 publications

(15 citation statements)

References 100 publications

(132 reference statements)

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“…To identify at genome-scale genes that, when deleted, lead to toxicity only in the presence of 3K inclusions, we turned to CRISPR/Cas9, a system that enables genome-scale deletion screens in human cells (Naxerova et al, 2021;Shalem et al, 2013). Advantageously, the pB system can be adapted easily to cell lines as discovery model amenable to high-throughput genome-wide pooled genetic screening.…”

Section: Convergence Of Genetic and Protein-protein Interaction Analy...mentioning

confidence: 99%

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Lam

Ndayisaba

Lewis

et al. 2022

Preprint

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In neurodegenerative proteinopathies, intracellular inclusions are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. Patient- derived iPSC models, while promising for disease modeling, do not form analogous inclusions in a reasonable timeframe and suffer from limited tractability and scalability. Here, we developed an iPSC toolbox that utilizes piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of misfolding-prone proteins. The system is scalable and amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of- principle, cortical neuron alpha-synuclein inclusionopathy models were engineered to form inclusions spontaneously or through exogenous seeding by alpha-synuclein fibrils. These models recapitulated known fibril- and lipid-rich inclusion subtypes in human brain, shedding light on their formation and consequences. Genetic-modifier and protein-interaction screens identified sequestered proteins in these inclusions, including RhoA, that were deleterious to cells when lost. This new iPSC platform should facilitate biological and drug discovery for neurodegenerative proteinopathies.

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Section: Convergence Of Genetic and Protein-protein Interaction Analy...mentioning

confidence: 99%

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Lam

Ndayisaba

Lewis

et al. 2022

Preprint

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“…Genome-scale CRISPR screens have emerged as a powerful tool to address this need. In addition to CRISPR-Cas9 screens that we and others have performed to identify genes involved in the step-wise differentiation from hPSCs to insulin-secreting β cells)( 115 , 116 , 117 , 118 ) recent studies have leveraged CRISPR screens to directly uncover immunomodulatory factors that mediate SCDI survival post-transplantation. For these experiments, a pool of cells is created where each cell has a different gene knocked out.…”

Section: Future Prospects: Genetic Screensmentioning

confidence: 99%

Genetic Engineering of Immune Evasive Stem Cell-Derived Islets

et al. 2022

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Genome editing has the potential to revolutionize many investigative and therapeutic strategies in biology and medicine. In the field of regenerative medicine, one of the leading applications of genome engineering technology is the generation of immune evasive pluripotent stem cell-derived somatic cells for transplantation. In particular, as more functional and therapeutically relevant human pluripotent stem cell-derived islets (SCDI) are produced in many labs and studied in clinical trials, there is keen interest in studying the immunogenicity of these cells and modulating allogeneic and autoimmune immune responses for therapeutic benefit. Significant experimental work has already suggested that elimination of Human Leukocytes Antigen (HLA) expression and overexpression of immunomodulatory genes can impact survival of a variety of pluripotent stem cell-derived somatic cell types. Limited work published to date focuses on stem cell-derived islets and work in a number of labs is ongoing. Rapid progress is occurring in the genome editing of human pluripotent stem cells and their progeny focused on evading destruction by the immune system in transplantation models, and while much research is still needed, there is no doubt the combined technologies of genome editing and stem cell therapy will profoundly impact transplantation medicine in the future.

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“…The transcription factors (TFs) OCT4 (POU5F1), NANOG, and SOX2 are well-established master regulators of pluripotent identity 1-5 that cooperatively regulate one another as well as a large set of downstream genes in hPSCs 6,7 . However, the downregulation of these master regulators during hPSC differentiation is accompanied by decreased susceptibility to apoptosis, with OCT4 depletion in some contexts even imparting a competitive advantage in hESC self-renewal 8,9 . In other words, these positive regulators of pluripotency are not always positive regulators of self-renewal.…”

Section: Introductionmentioning

confidence: 99%

“…Recent advances in CRISPR-Cas9 genome editing have enabled the use of large-scale loss-offunction screens to interrogate self-renewal through assaying cell survival and proliferation [9][10][11][12][13] .…”

Section: Introductionmentioning

confidence: 99%

Parallel genome-scale CRISPR screens distinguish pluripotency and self-renewal

Rosen

Cho

et al. 2023

Preprint

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SummaryPluripotent stem cells are defined by both the ability to unlimitedly self-renew and differentiate to any somatic cell lineage, but understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. We performed four parallel genome-scale CRISPR-Cas9 screens to investigate the interplay between these two aspects of pluripotency. Our comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including many mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control stem cell identity. We further discovered a core set of factors that control both stem cell fitness and pluripotency identity, including an interconnected network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus self-renewal, and offer a valuable model for categorizing gene function in broad biological contexts.

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Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior

Cited by 18 publications

References 100 publications

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Genetic Engineering of Immune Evasive Stem Cell-Derived Islets

Parallel genome-scale CRISPR screens distinguish pluripotency and self-renewal

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