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

Pluripotent 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.

[1]  C. Leslie,et al.  CRISPR screening uncovers a central requirement for HHEX in pancreatic lineage commitment and plasticity restriction , 2022, Nature Cell Biology.

[2]  S. Elledge,et al.  Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior , 2021, Genes & development.

[3]  R. Jaenisch,et al.  OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human , 2021, Nature Communications.

[4]  L. Aaltonen,et al.  Human cell transformation by combined lineage conversion and oncogene expression , 2021, Oncogene.

[5]  O. Elemento,et al.  QSER1 protects DNA methylation valleys from de novo methylation , 2021, Science.

[6]  Joshua M. Dempster,et al.  Chronos: a CRISPR cell population dynamics model , 2021, bioRxiv.

[7]  Ellen F. Macnamara,et al.  Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation , 2021, Science Advances.

[8]  S. Sidoli,et al.  Guide for protein fold change and p-value calculation for non-experts in proteomics. , 2020, Molecular omics.

[9]  Joshua M. Dempster,et al.  Integrated cross-study datasets of genetic dependencies in cancer , 2020, Nature Communications.

[10]  Michael A. Beer,et al.  Enhancer Predictions and Genome-Wide Regulatory Circuits. , 2020, Annual review of genomics and human genetics.

[11]  Médecine,et al.  Tumor Suppressor Gene , 2010 .

[12]  R. Blelloch,et al.  Epigenetic control of transcriptional regulation in pluripotency and early differentiation , 2019, Development.

[13]  Danwei Huangfu,et al.  Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency , 2019, Wiley interdisciplinary reviews. Systems biology and medicine.

[14]  Aviad Tsherniak,et al.  Extracting Biological Insights from the Project Achilles Genome-Scale CRISPR Screens in Cancer Cell Lines , 2019, bioRxiv.

[15]  Christina S. Leslie,et al.  FOXA2 Is Required for Enhancer Priming during Pancreatic Differentiation , 2019, Cell reports.

[16]  N. Benvenisty,et al.  Defining Human Pluripotency. , 2019, Cell stem cell.

[17]  Matthew C. Canver,et al.  TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network. , 2019, Molecular cell.

[18]  J. Moffat,et al.  Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies. , 2019, Cell reports.

[19]  Alireza Hadj Khodabakhshi,et al.  Metascape provides a biologist-oriented resource for the analysis of systems-level datasets , 2019, Nature Communications.

[20]  M. Beer,et al.  Genome-scale screens identify JNK/JUN signaling as a barrier for pluripotency exit and endoderm differentiation , 2019, Nature Genetics.

[21]  Robert C. Altshuler,et al.  Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes. , 2019, Cell Reports.

[22]  Matthew C. Canver,et al.  CRISPResso2 provides accurate and rapid genome editing sequence analysis , 2019, Nature Biotechnology.

[23]  Y. Kee,et al.  The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin , 2018, Nucleic acids research.

[24]  Jesse R. Dixon,et al.  A non-canonical BRD9-containing BAF chromatin remodeling complex regulates naive pluripotency in mouse embryonic stem cells , 2018, Nature Communications.

[25]  Damian Szklarczyk,et al.  STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets , 2018, Nucleic Acids Res..

[26]  J. Ranish,et al.  Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes , 2018, Cell.

[27]  C. Lareau,et al.  A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation , 2018, Nature Cell Biology.

[28]  N. Benvenisty,et al.  Defining essential genes for human pluripotent stem cells by CRISPR–Cas9 screening in haploid cells , 2018, Nature Cell Biology.

[29]  Y. Seki.  PRDM14 Is a Unique Epigenetic Regulator Stabilizing Transcriptional Networks for Pluripotency , 2018, Front. Cell Dev. Biol..

[30]  Nipun Verma,et al.  CRISPR/Cas9-Based Engineering of the Epigenome. , 2017, Cell stem cell.

[31]  G. Pan,et al.  PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs , 2017, Nature Communications.

[32]  Daesik Kim,et al.  Genome editing reveals a role for OCT4 in human embryogenesis , 2017, Nature.

[33]  L. Studer,et al.  A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages. , 2017, Cell stem cell.

[34]  Phillip G. Montgomery,et al.  Defining a Cancer Dependency Map , 2017, Cell.

[35]  Ann E. Sizemore,et al.  Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells , 2017, Nature Genetics.

[36]  D. Kent,et al.  Proliferation Drives Aging-Related Functional Decline in a Subpopulation of the Hematopoietic Stem Cell Compartment , 2017, Cell reports.

[37]  T. Evans,et al.  Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development. , 2017, Cell stem cell.

[38]  Alexander Lex,et al.  UpSetR: an R package for the visualization of intersecting sets and their properties , 2017, bioRxiv.

[39]  I. Amit,et al.  Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq , 2016, Cell.

[40]  Thomas M. Norman,et al.  A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response , 2016, Cell.

[41]  Thomas M. Norman,et al.  Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens , 2016, Cell.

[42]  André F. Rendeiro,et al.  Pooled CRISPR screening with single-cell transcriptome read-out , 2017, Nature Methods.

[43]  Wenjun Guo,et al.  Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation. , 2016, Cell reports.

[44]  J. Collins,et al.  RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming. , 2016, Cell reports.

[45]  S. Sayols,et al.  Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation , 2016, Cell cycle.

[46]  Guangchuang Yu,et al.  ReactomePA: an R/Bioconductor package for reactome pathway analysis and visualization. , 2016, Molecular bioSystems.

[47]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[48]  J. Moffat,et al.  BAGEL: a computational framework for identifying essential genes from pooled library screens , 2015, bioRxiv.

[49]  B. Blencowe,et al.  Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming , 2015, Genes & development.

[50]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[51]  Hanspeter Pfister,et al.  UpSet: Visualization of Intersecting Sets , 2014, IEEE Transactions on Visualization and Computer Graphics.

[52]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[53]  R. Mailman,et al.  Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells , 2014, Stem cell reports.

[54]  Zengrong Zhu,et al.  An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. , 2014, Cell stem cell.

[55]  T. Rana,et al.  Genome-wide functional analysis reveals factors needed at the transition steps of induced reprogramming. , 2014, Cell reports.

[56]  Neville E. Sanjana,et al.  Improved vectors and genome-wide libraries for CRISPR screening , 2014, Nature Methods.

[57]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[58]  A. Letai,et al.  High mitochondrial priming sensitizes hESCs to DNA-damage-induced apoptosis. , 2013, Cell stem cell.

[59]  K. Shirahige,et al.  PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. , 2013, Cell stem cell.

[60]  Yu-Hsiang Lin,et al.  The EP300, KDM5A, KDM6A and KDM6B Chromatin Regulators Cooperate with KLF4 in the Transcriptional Activation of POU5F1 , 2012, PloS one.

[61]  V. Dixit,et al.  Phosphorylation-dependent activity of the deubiquitinase DUBA , 2012, Nature Structural &Molecular Biology.

[62]  Bing Lim,et al.  A precarious balance: pluripotency factors as lineage specifiers. , 2011, Cell stem cell.

[63]  Jennifer M. Bolin,et al.  Chemically defined conditions for human iPS cell derivation and culture , 2011, Nature Methods.

[64]  N. D. Clarke,et al.  A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity , 2010, Nature.

[65]  J. Rossant,et al.  Phenotypic annotation of the mouse X chromosome. , 2010, Genome research.

[66]  Alexey I Nesvizhskii,et al.  An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency , 2009, Proceedings of the National Academy of Sciences.

[67]  M. Tomishima,et al.  Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling , 2009, Nature Biotechnology.

[68]  Benjamin L. Kidder,et al.  SWI/SNF‐Brg1 Regulates Self‐Renewal and Occupies Core Pluripotency‐Related Genes in Embryonic Stem Cells , 2009, Stem cells.

[69]  U. Hofmann,et al.  Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse , 2008, Development.

[70]  E. Pietras,et al.  A Deubiquitinase That Regulates Type I Interferon Production , 2007, Science.

[71]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[72]  T. Perlmann,et al.  The establishment of neuronal properties is controlled by Sox4 and Sox11. , 2006, Genes & development.

[73]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[74]  X. Chen,et al.  The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells , 2006, Nature Genetics.

[75]  E. Kroon,et al.  Efficient differentiation of human embryonic stem cells to definitive endoderm , 2005, Nature Biotechnology.

[76]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[77]  Megan F. Cole,et al.  Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells , 2005, Cell.

[78]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[79]  M. Daly,et al.  PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes , 2003, Nature Genetics.

[80]  M. Murakami,et al.  The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells , 2003, Cell.

[81]  J. Nichols,et al.  Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells , 2003, Cell.

[82]  William C Hahn,et al.  Lentivirus-delivered stable gene silencing by RNAi in primary cells. , 2003, RNA.

[83]  J. Rossant,et al.  FoxH1 (Fast) functions to specify the anterior primitive streak in the mouse. , 2001, Genes & development.

[84]  P. Hoodless,et al.  Formation of the definitive endoderm in mouse is a Smad2-dependent process. , 2000, Development.

[85]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[86]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[87]  H. Schöler,et al.  Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4 , 1998, Cell.

[88]  G. Martin,et al.  Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[89]  M. Kaufman,et al.  Establishment in culture of pluripotential cells from mouse embryos , 1981, Nature.

[90]  Cedric E. Ginestet.  ggplot2: Elegant Graphics for Data Analysis , 2011 .

[91]  J. Rossant,et al.  The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. , 1998, Genes & development.