The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance1,2 that can confound analyses and undermine reproducibility3. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

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  1. 1.

    & Origins and implications of pluripotent stem cell variability and heterogeneity. Nat. Rev. Mol. Cell Biol. 14, 357–368 (2013)

  2. 2.

    et al. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas. Cell Res. 26, 304–319 (2016)

  3. 3.

    & Drug development: raise standards for preclinical cancer research. Nature 483, 531–533 (2012)

  4. 4.

    , , , & Mouse ENU mutagenesis. Hum. Mol. Genet. 8, 1955–1963 (1999)

  5. 5.

    , , & Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445–448 (1986)

  6. 6.

    et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001)

  7. 7.

    , & A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002)

  8. 8.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  9. 9.

    et al. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013)

  10. 10.

    , , & Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016)

  11. 11.

    et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016)

  12. 12.

    et al. Genetic background drives transcriptional variation in human induced pluripotent stem cells. PLoS Genet. 10, e1004432 (2014)

  13. 13.

    , & A comparison of the germline potential of differently aged ES cell lines and their transfected descendants. Transgenic Res. 6, 223–231 (1997)

  14. 14.

    et al. Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3, 777–779 (2006)

  15. 15.

    et al. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc. Natl Acad. Sci. USA 102, 7221–7226 (2005)

  16. 16.

    et al. Enhanced gene trapping in mouse embryonic stem cells. Nucleic Acids Res. 36, e133 (2008)

  17. 17.

    et al. Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells. Nucleic Acids Res. 40, e97 (2012)

  18. 18.

    , , & Molecular reconstruction of sleeping beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510 (1997)

  19. 19.

    et al. The Drosophila gene disruption project: progress using transposons with distinctive site specificities. Genetics 188, 731–743 (2011)

  20. 20.

    et al. Transposon-mediated genome manipulation in vertebrates. Nat. Methods 6, 415–422 (2009)

  21. 21.

    et al. An α-E-catenin gene trap mutation defines its function in preimplantation development. Proc. Natl Acad. Sci. USA 94, 901–906 (1997)

  22. 22.

    , , & Species-specific receptor recognition by a minor-group human rhinovirus (HRV): HRV serotype 1A distinguishes between the murine and the human low-density lipoprotein receptor. J. Virol. 76, 6957–6965 (2002)

  23. 23.

    et al. Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus. Proc. Natl Acad. Sci. USA 91, 1839–1842 (1994)

  24. 24.

    et al. AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency. Nat. Med. 15, 159–168 (2009)

  25. 25.

    , , , & Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA). J. Biol. Chem. 283, 25428–25436 (2008)

  26. 26.

    et al. Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis. Cell Death Differ. 11, 1009–1016 (2004)

  27. 27.

    et al. Interaction of phospholipase A/acyltransferase-3 with Pex19p: a possible involvement in the down-regulation of peroxisomes. J. Biol. Chem. 290, 17520–17534 (2015)

  28. 28.

    et al. PLA2G16 represents a switch between entry and clearance of Picornaviridae. Nature 541, 412–416 (2017)

  29. 29.

    , & Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887 (2011)

  30. 30.

    et al. Identification and functional analysis of endothelial tip cell-enriched genes. Blood 116, 4025–4033 (2010)

  31. 31.

    , & Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 115, 5102–5110 (2010)

  32. 32.

    et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell Biol. 12, 943–953 (2010)

  33. 33.

    et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007)

  34. 34.

    , , , & Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development. Mol. Reprod. Dev. 30, 18–26 (1991)

  35. 35.

    , , & Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010)

  36. 36.

    , , & Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery. Am. J. Physiol. Heart Circ. Physiol. 280, H2441–H2450 (2001)

  37. 37.

    et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010)

  38. 38.

    1,500 scientists lift the lid on reproducibility. Nature 533, 452–454 (2016)

  39. 39.

    et al. Haplobank methods collection. Protoc. Exch. (2017)

  40. 40.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  41. 41.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)

  42. 42.

    , , & Karyotyping mouse chromosomes by multiplex-FISH (M-FISH). Chromosome Res. 9, 211–214 (2001)

  43. 43.

    & Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

  44. 44.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

  45. 45.

    , , , & Improving RNA-seq expression estimates by correcting for fragment bias. Genome Biol. 12, R22 (2011)

  46. 46.

    et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008)

  47. 47.

    , , & ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9 (2015)

  48. 48.

    , & Intravenous injections in neonatal mice. J. Vis. Exp. 93, e52037 (2014)

  49. 49.

    et al. Structural basis for the acyltransferase activity of lecithin:retinol acyltransferase-like proteins. J. Biol. Chem. 287, 23790–23807 (2012)

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We thank all members of our laboratories, IMBA/IMP and VBCF services for support and Life Science Editors for assistance; B. Knapp, I. Filipuzzi and T. Aust for clone picking, N. R. Movva and T. Bouwmeester (NIBR) for support, and K. Handler for the differentiation protocols. The Haplobank is funded by the Austrian National Bank (OeNB), an Advanced ERC grant and Era of Hope/National Coalition against Breast Cancer/DoD (to J.M.P.). U.E. is a Wittgenstein Prize fellow. D.B. is supported by FWF P23308-B13. A.S. is supported by an ERC Consolidator Grant, Boehringer Ingelheim and FFG.

Author information

Author notes

    • Ulrich Elling
    •  & Reiner A. Wimmer

    These authors contributed equally to this work.


  1. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria

    • Ulrich Elling
    • , Reiner A. Wimmer
    • , Andreas Leibbrandt
    • , Thomas Burkard
    • , Georg Michlits
    • , Alexandra Leopoldi
    • , Dana Abdeen
    • , Sergei Zhuk
    • , Cornelia Handl
    • , Julia Liebergesell
    • , Maria Hubmann
    • , Anna-Maria Husa
    • , Manuela Kinzer
    • , Nicole Schuller
    • , Ellen Wetzel
    • , Nina van de Loo
    • , Jorge Arturo Zepeda Martinez
    • , Chukwuma A. Agu
    • , Oliver Bell
    •  & Josef M. Penninger
  2. Vienna Biocenter Core Facilities, Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria

    • Thomas Micheler
  3. MRC Laboratory for Molecular Cell Biology and Institute for the Physics of Living Systems, University College London, London, UK

    • Irene M. Aspalter
  4. Novartis Institutes for BioMedical Research, Basel, Switzerland

    • David Estoppey
    • , Ralph Riedl
    •  & Dominic Hoepfner
  5. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    • Fengtang Yang
    •  & Beiyuan Fu
  6. Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr Gasse 9, Vienna, Austria

    • Thomas Dechat
    •  & Dieter Blaas
  7. Paul Ehrlich Institut, Paul Ehrlich Strasse 51–59, 63225 Langen, Germany

    • Zoltán Ivics
  8. Max-Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

    • Holger Gerhardt
  9. German Center for Cardiovascular Research, Berlin, Germany

    • Holger Gerhardt
  10. Berlin Institute of Health, Berlin, Germany

    • Holger Gerhardt
  11. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Dr. Bohr Gasse 7, 1030 Vienna, Austria

    • Alexander Stark


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U.E. generated the haploid library with technical support from A.Lei., C.H., J.L., M.H., A.-M.H., M.K., N.S., E.W., N.v.d.L., D.H., R.R. and D.E. U.E., R.A.W. and A.Leo. characterized cell lines. A.Lei., G.M., U.E., D.B. and T.D. performed rhinovirus work. A.S., T.B. and T.M. wrote the bioinformatics algorithms and set up the Haplobank website. S.Z. performed RACE experiments, F.Y. and B.F. performed karyotyping experiments and C.A.A. supported standardization. J.A.Z.M. and O.B. performed ATAC-sequencing. Z.I. advized on mutagenesis vectors. R.A.W., I.M.A., D.A., A.Leo. and H.G. performed blood vessel experiments. U.E. and J.M.P. coordinated the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ulrich Elling or Josef M. Penninger.

Reviewer Information Nature thanks S. Narumiya and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Reporting Summary

  2. 2.

    Supplementary Figures

    This file contains full uncropped scans of DNA gels and Western blots used in Extended Data Figure 8.

  3. 3.

    Supplementary Table

    This table shows the numbers of clones available with respect to different mutagens, orientation of the inserted gene trap to gene transcription, as well as the number of different genes hit. A gene is defined as the genomic region between the transcriptional start and stop sites. www.haplobank.at

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