Human pluripotent reprogramming with CRISPR activators

Weltner, Jere; Balboa, Diego; Katayama, Shintaro; Bespalov, Maxim M.; Krjutškov, Kaarel; Jouhilahti, Eeva-Mari; Trokovic, Ras; Kere, Juha; Otonkoski, Timo

doi:10.1038/s41467-018-05067-x

Cited by 146 publications

(121 citation statements)

References 52 publications

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“…We designed guide RNAs for the LEUTX promoter and putative novel enhancer regions (Extended Data Fig. 5e) to experimentally test these using the CRISPRa activation system 19 in HEK293 cells. The expression level of LEUTX nearly doubled when the guide RNAs targeting the promoter region were transfected together with the putative enhancer 1 targeting guide RNAs in comparison to the promoter targeting guide RNAs only (Fig.…”

Section: Resultsmentioning

confidence: 99%

DUX4 regulates oocyte to embryo transition in human

Vuoristo

Hydén-Granskog

Yoshihara

et al. 2019

Preprint

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45During the human oocyte-to-embryo transition, the fertilized oocyte undergoes 46 final maturation and the embryo genome is gradually activated during the first 47 three cell divisions. How this transition is coordinated in humans is largely 48 unknown. We show that the double homeodomain transcription factor DUX4 49 contributes to this transition. DUX4 knockdown in human zygotes caused 50 insufficient transcriptome reprogramming as observed three days after 51 fertilization. Induced DUX4 expression in human embryonic stem cells activated 52 transcription of thousands of newly identified bi-directional transcripts, including 53 putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 54 protein interacted with transcriptional modifiers that are known to couple 55 enhancers and promoters. Taken together, our results reveal that DUX4 is a 56 pioneer regulating oocyte-to-embryo transition in human through activation of 57 intergenic genome, especially enhancers, and hence setting the stage for early 58 human embryo development. 59 60 61 62 63 64 65 4 Mammalian pre-implantation development commences with oocyte-to-embryo 66 transition, which involves fundamental changes in the epigenetic landscapes, 67 modulation of cell cycle control, and translation or clearance of selected maternal 68 mRNAs, culminating to embryonic genome activation 1,2 . The pioneer regulators 69 orchestrating the oocyte-to-embryo transition and first embryo genome activation steps 70 in human remain poorly understood. The conserved double homeodomain transcription 71factor DUX4 represents a plausible candidate regulating the oocyte-to-embryo 72 transition in humans, given its capacity to activate germline genes and genomic repeat 73 elements 3-5 . Here we show that DUX4 is able to launch the first reprogramming steps 74 from oocyte to embryo in human by activating thousands of novel enhancers and 75 therein, modulating the transcriptome and chromatin. Human DUX4 knockdown 76 embryos are viable until the third day of development, but their transcriptome is 77 severely altered. Our proteomics approaches suggest that DUX4 binds to Mediator 78 complex and chromatin modifiers through its C-terminal domains, providing a likely 79 explanation to how DUX4 may extensively modulate the genome. This study implies a 80 wider role for DUX4 as a cellular gate keeper acting both as a general genomic modifier 81 of cell fate as well as a specific inducer of first wave embryo genome activation genes. 82 83Results 84 Quantification of DUX4 in human embryos 85The DUX4 induced gene network is highly conserved 6 and recent reports showed that 86 DUX4 is expressed in early human embryos 3,4 . However, details of this dynamic 87 process, including initiation of DUX4 expression, remained ambiguous. Therefore, we 88Given the short-term and precise manifestation of DUX4 mRNA and protein in human 107 zygotes and early cleavage stage embryos, we next asked how DUX4 regulates the first 108 steps of human embryo development. We microinjected either DUX4...

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Section: Resultsmentioning

confidence: 99%

DUX4 regulates oocyte to embryo transition in human

Vuoristo

Hydén-Granskog

Yoshihara

et al. 2019

Preprint

Self Cite

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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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“…Two components of the oriP and Epstein-Barr nuclear antigen-1 (EBNA-1) are also used widely in episomal plasmids [32][33][34][35][36][37][38] . Yu et al reported that human iPSCs that are completely free of plasmids and transgene sequences could be derived from fibroblasts by a single transfection with oriP/ EBNA-1-based episomal plasmids [39][40][41][42] .…”

Section: Non-integrative Methods Of Ipsc Productionmentioning

confidence: 99%

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

Wang

Loh

2019

Cell Transplant

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The term episomal induced pluripotent stem cells (EiPSCs) refers to somatic cells that are reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrative episomal vector methods. This reprogramming process has a better safety profile compared with integrative methods using viruses. There is a current trend toward using episomal plasmid reprogramming to generate iPSCs because of the improved safety profile. Clinical reports of potential human cell sources that have been successfully reprogrammed into EiPSCs are increasing, but no review or summary has been published. The functional applications of EiPSCs and their potential uses in various conditions have been described, and these may be applicable to clinical scenarios. This review summarizes the current direction of EiPSC research and the properties of these cells with the aim of explaining their potential role in clinical applications and functional restoration.

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Human pluripotent reprogramming with CRISPR activators

Cited by 146 publications

References 52 publications

DUX4 regulates oocyte to embryo transition in human

DUX4 regulates oocyte to embryo transition in human

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

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

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