CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency

Liu, Peng; Chen, Meng; Liu, Yanxia; Qi, Lei S.; Ding, Sheng

doi:10.1016/j.stem.2017.12.001

Cited by 142 publications

(98 citation statements)

References 32 publications

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“…CRISPR-activation (CRISPRa) is a potent tool for selective transcriptional upregulation of endogenous genes, which functions by targeting a dead Cas9 (dCas9) with transcriptional co-activators to gene promoters using short-guide RNAs (sgRNAs) (Cheng et al 2013.;Gilbert et al 2014;Chavez et al 2015;Konermann et al 2014), and has been successfully used for cellular reprogramming and the study of cellular transitions (Chakraborty et al 2014;Black et al 2016;Peng Liu et al 2018;Weltner et al 2018;Yang et al 2019;Genga et al 2019). CRISPRa is preferable to traditional overexpression techniques, such as cloned cDNA overexpression, as it leads to target gene activation at physiologicallyrelevant levels (Chavez et al 2015;Sanson et al 2018;Yang et al 2019).…”

Section: Introductionmentioning

confidence: 99%

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Alda-Catalinas

Bredikhin

Hernando-Herraez

et al. 2019

Preprint

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Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming processes and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics to systematically probe candidate regulators of ZGA. We screened 230 epigenetic and transcriptional regulators by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNAsequencing (scRNA-seq) of CRISPRa-perturbed cells, we generated approximately 200,000 single-cell transcriptomes, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using integrative dimensionality reduction of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 44 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. Upon upregulation of these factors, including the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, ESCs adopt an early embryonic-like state. Supporting their roles as ZGA regulators, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes, however, overexpression of Dppa2 in Smarca5 knock-out ESCs, but not vice versa, rescues ZGA-like expression, suggesting that Smarca5 regulates ZGA upstream and via Dppa2. Together, our single-cell transcriptomic profiling of CRISPRaperturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA. Highlights• First large-scale screen combining pooled CRISPRa with scRNA-seq. • Multi-omics factor analysis identifies a ZGA-like signature for 44 of the candidate regulators. • Dppa2, Smarca5 and Patz1 were validated as strong inducers of ZGA gene expression. • Smarca5 regulates zygotic genome activation in a Dppa2-dependent manner.

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Section: Introductionmentioning

confidence: 99%

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Alda-Catalinas

Bredikhin

Hernando-Herraez

et al. 2019

Preprint

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“…With other approaches, for example using nonintegrating vectors [104,105] , introduction of stemness proteins [106,107] , or pharmaceutically with a suitable combination of small molecules, have been developed to address this problem to a certain extent [44,108,109] . Recently, a new method to generate iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci shed light on how targeted chromatin remodeling triggers pluripotency induction [110] . Authentic porcine iPSCs cannot survive in culture following the silencing or downregulation of the reprogramming factors.…”

Section: Progress On Inducing Pluripotent Stem Cells Of Pig and Othermentioning

confidence: 99%

Porcine pluripotent stem cells: progress, challenges and prospects

Han¹,

Miao²,

Hua³

et al. 2019

Front. Agr. Sci. Eng.

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Pluripotent stem cells (PSCs) are characterized by their capacity for high self-renewal and multiple differentiation potential and include embryonic stem cells, embryonic germ cells and induced PSCs. PSCs provide a very suitable model for the studies of human diseases, drugs screening, regenerative medicine and developmental biology research. Pigs are considered as an ideal model for preclinical development of human xenotransplantation, therapeutic approaches and regenerative medicine because of their size and physiological similarity to humans. However, lack of knowledge about the derivation, characterization and pluripotency mechanisms of porcine PSCs hinders progress in these biotechnologies. In this review, we discuss the latest progress on porcine PSCs generation, evaluation criteria for pluripotency, the scientific and technical questions arising from these studies. We also introduce our perspectives on porcine PSC research, in the hope of providing new ideas for generating naive porcine PSCs and animal breeding.

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“…The phenotypic discordance of monozygotic twins, most likely due to epigenetic differences, became more noticeable with age. Fourthly, epigenome editing and transcriptional alteration are sufficient to induce pluripotency (Liu et al 2018a), differentiation into a specific cell type or direct conversion between cell types (Black et al 2016) without the need to use exogenous reprogramming factors or transgene integration into the host genome (Takahashi et al 2007; Takahashi and Yamanaka 2006). Epigenome editing enabled rapid epigenetic remodeling and sustained transcriptional activation of endogenous master transcription factors.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

In vivo epigenome editing and transcriptional modulation using CRISPR technology

Lau

Suh

2018

Transgenic Res

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The rapid advancement of CRISPR technology has enabled targeted epigenome editing and transcriptional modulation in the native chromatin context. However, only a few studies have reported the successful editing of the epigenome in adult animals in contrast to the rapidly growing number of in vivo genome editing over the past few years. In this review, we discuss the challenges facing in vivo epigenome editing and new strategies to overcome the huddles. The biggest challenge has been the difficulty in packaging dCas9 fusion proteins required for manipulation of epigenome into the adeno-associated virus (AAV) delivery vehicle. We review the strategies to address the AAV packaging issue, including small dCas9 orthologues, truncated dCas9 mutants, a split-dCas9 system, and potent truncated effector domains. We discuss the dCas9 conjugation strategies to recruit endogenous chromatin modifiers and remodelers to specific genomic loci, and recently developed methods to recruit multiple copies of the dCas9 fusion protein, or to simultaneous express multiple gRNAs for robust epigenome editing or synergistic transcriptional modulation. The use of Cre-inducible dCas9-expressing mice or a genetic cross between dCas9- and sgRNA-expressing flies has also helped overcome the transgene delivery issue. We provide perspective on how a combination use of these strategies can facilitate in vivo epigenome editing and transcriptional modulation.

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CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency

Cited by 142 publications

References 32 publications

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Porcine pluripotent stem cells: progress, challenges and prospects

In vivo epigenome editing and transcriptional modulation using CRISPR technology

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