CRISPR screening uncovers a central requirement for HHEX in pancreatic lineage commitment and plasticity restriction

Yang, Dapeng; Cho, Hyunwoo; Tayyebi, Zakieh; Shukla, Abhijit; Luo, Renhe; Dixon, Gary; Ursu, Valeria; Stransky, Stephanie; Tremmel, Daniel M.; Sackett, Sara Dutton; Koche, Richard P.; Kaplan, Samuel J.; Li, Qing V.; Park, Jiwoon; Zhu, Zuoyan; Rosen, B.P.; Pulecio, Julián; Shi, Zhong‐Dong; Bram, Yaron; Schwartz, Robert E.; Odorico, Jon S.; Wright, Christopher V.E.; Leslie, Christina; Huangfu, Danwei

doi:10.1038/s41556-022-00946-4

Cited by 21 publications

(39 citation statements)

References 84 publications

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“…scRNA-seq was performed as previously described 58 . Cells were harvested every 12h during DE differentiation for scRNA-seq experiments with targeted collection ranging from 3000 to 6000 cells.…”

Section: Methodsmentioning

confidence: 99%

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Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Luo

Yan

et al. 2023

Preprint

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Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

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Section: Methodsmentioning

confidence: 99%

“…Y-27632. Cells were counted by using Vi-CELL XR Cell Viability Analyzer (Beckman Coulter), and scRNA-seq scRNA-seq was performed as previously described58 . Cells were harvested every 12h during DE differentiation for scRNA-seq experiments with targeted collection ranging from 3000 to 6000 cells.…”

mentioning

confidence: 99%

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Luo

Yan

et al. 2023

Preprint

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“…We next conducted parallel screens in NE and DE differentiation conditions to distinguish whether the regulation of pluripotency was largely differentiation context dependent, or if a shared group of regulators might control the dissolution of pluripotency regardless of differentiation context. Using a pooled screening strategy 17,21,22 , we infected H1 OCT4 GFP/+ iCas9 hESCs with a genome-scale CRISPR library 23 (Figure 1A). OCT4-GFP hi and OCT4-GFP lo were isolated by fluorescence-activated cell sorting (FACS) on day 1.5 of NE (Figure S2A,B) and day 2.5 of DE (Figure S2C,D) differentiations, matching the timepoints when OCT4-GFP was rapidly downregulated (Figure S1C).…”

Section: Genome-scale Screens For Regulators Of Pluripotencymentioning

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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“…Since SCislets originate from stem cells, they can be genetically modified by introducing specific mutations into wild-type hESCs or hiPSCs, as well as correcting mutations or disease-associated gene variants in hiPSCs generated from diabetic patients. A plethora of diabetes-associated genes have already been modelled by combining the use of SC-islets and CRISPR genome editing (59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73). Even for a complex disease like T1D, the possibility to model the effect of T1D risk and protective genes or single nucleotide polymorphisms on b-cell development and T-cell-mediated cytotoxicity has become feasible (74).…”

Section: Stem-cell-derived Islets (Sc-islets)mentioning

confidence: 99%

In vitro beta-cell killing models using immune cells and human pluripotent stem cell-derived islets: Challenges and opportunities

et al. 2023

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Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8+ T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop in vitro β-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of in vitro expanded CD8+ T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the β-cell/immune crosstalk in an islet microenvironment; the features that make β-cells more sensitive to autoimmunity; therapeutic agents that may modulate β-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor.

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CRISPR screening uncovers a central requirement for HHEX in pancreatic lineage commitment and plasticity restriction

Cited by 21 publications

References 84 publications

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

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

In vitro beta-cell killing models using immune cells and human pluripotent stem cell-derived islets: Challenges and opportunities

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