Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library

Nature Biotechnology volume 32, pages 267–273 (2014)Cite this article

Subjects

Abstract

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Stable expression of Cas9 and gRNA from single-copy transgenes can induce site-specific DSBs.
Figure 2: Lentiviral delivery of gRNA expression cassettes.
Figure 3: Analyses of 52 gRNAs targeting 26 genes involved in the GPI-anchor biosynthesis pathway.
Figure 4: Generation of a mouse genome-wide lentiviral gRNA library.
Figure 5: Genetic screens using the genome-wide gRNA library and genetic validation assays of the novel candidate genes.

Similar content being viewed by others

Accession codes

Accessions

European Nucleotide Archive

References

  1. Forsburg, S.L. The art and design of genetic screens: yeast. Nat. Rev. Genet. 2, 659–668 (2001).

    Article  CAS  Google Scholar 

  2. Jorgensen, E.M. & Mango, S.E. The art and design of genetic screens: Caenohabditis elegans. Nat. Rev. Genet. 3, 356–369 (2002).

    Article  CAS  Google Scholar 

  3. Boutros, M. & Ahringer, J. The art and design of genetic screens: RNA interference. Nat. Rev. Genet. 9, 554–566 (2008).

    Article  CAS  Google Scholar 

  4. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  Google Scholar 

  5. Iorns, E., Lord, C.J., Turner, N. & Ashworth, A. Utilizing RNA interference to enhance cancer drug discovery. Nat. Rev. Drug Discov. 6, 556–568 (2007).

    Article  CAS  Google Scholar 

  6. Carette, J.E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).

    Article  CAS  Google Scholar 

  7. Carette, J.E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nat. Biotechnol. 29, 542–546 (2011).

    Article  CAS  Google Scholar 

  8. Carette, J.E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011).

    Article  CAS  Google Scholar 

  9. Leeb, M. & Wutz, A. Derivation of haploid embryonic stem cells from mouse embryos. Nature 479, 131–134 (2011).

    Article  CAS  Google Scholar 

  10. Yang, H. et al. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149, 605–617 (2012).

    Article  CAS  Google Scholar 

  11. Elling, U. et al. Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9, 563–574 (2011).

    Article  CAS  Google Scholar 

  12. Yang, H. et al. Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes. Cell Res. 23, 1187–1200 (2013).

    Article  CAS  Google Scholar 

  13. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  Google Scholar 

  14. Joung, J.K. & Sander, J.D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).

    Article  CAS  Google Scholar 

  15. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  16. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  17. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  18. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  Google Scholar 

  19. Takeda, J. et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 73, 703–711 (1993).

    Article  CAS  Google Scholar 

  20. Kinoshita, T., Fujita, M. & Maeda, Y. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J. Biochem. 144, 287–294 (2008).

    Article  CAS  Google Scholar 

  21. Gordon, V.M. et al. Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors. J. Biol. Chem. 274, 27274–27280 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  24. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).

    Article  CAS  Google Scholar 

  25. Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

    Article  Google Scholar 

  26. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  CAS  Google Scholar 

  27. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).

    Article  CAS  Google Scholar 

  28. Hakem, R. et al. The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell 85, 1009–1023 (1996).

    Article  CAS  Google Scholar 

  29. Tsuzuki, T. et al. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA 93, 6236–6240 (1996).

    Article  CAS  Google Scholar 

  30. Luo, J. et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137, 835–848 (2009).

    Article  CAS  Google Scholar 

  31. Agaisse, H. et al. Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309, 1248–1251 (2005).

    Article  CAS  Google Scholar 

  32. Karlas, A. et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463, 818–822 (2010).

    Article  CAS  Google Scholar 

  33. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  34. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  Google Scholar 

  35. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  Google Scholar 

  36. Cadinanos, J. & Bradley, A. Generation of an inducible and optimized piggyBac transposon system. Nucleic Acids Res. 35, e87 (2007).

    Article  Google Scholar 

  37. Yusa, K., Rad, R., Takeda, J. & Bradley, A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat. Methods 6, 363–369 (2009).

    Article  CAS  Google Scholar 

  38. Carey, B.W. et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl. Acad. Sci. USA 106, 157–162 (2009).

    Article  CAS  Google Scholar 

  39. Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116–1124 (2008).

    Article  CAS  Google Scholar 

  40. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

  41. Pettitt, S.J. et al. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat. Methods 6, 493–495 (2009).

    Article  CAS  Google Scholar 

  42. Wang, W., Bradley, A. & Huang, Y. A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells. Genome Res. 19, 667–673 (2009).

    Article  CAS  Google Scholar 

  43. Abuin, A., Zhang, H. & Bradley, A. Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations. Mol. Cell. Biol. 20, 149–157 (2000).

    Article  CAS  Google Scholar 

  44. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  45. Quail, M.A. et al. Optimal enzymes for amplifying sequencing libraries. Nat. Methods 9, 10–11 (2012).

    Article  CAS  Google Scholar 

  46. Huang, da W. et al. DAVID gene ID conversion tool. Bioinformation 2, 428–430 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

We thank A. Bradley for comments on the manuscript, B. Ng and W. Cheng for the flow cytometry analyses, and the Sanger Institute DNA pipeline for the sequence analyses. We also thank J. Takeda and T. Kinoshita for providing us alpha-toxin and the cDNA expression vectors, respectively. Y.L. is supported by the Wellcome Trust PhD program. M.D.C.V.-H. is supported by the Cancer Research UK and Wellcome Trust PhD program. This work was supported by Wellcome Trust (WT077187). The mouse CRISPR library is available through Addgene. The plasmid DNAs are available at the Wellcome Trust Sanger Institute Archives (http://www.sanger.ac.uk/technology/clonerequests/).

Author information

Author notes

  1. Hiroko Koike-Yusa and Yilong Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK

    Hiroko Koike-Yusa, Yilong Li, E-Pien Tan, Martin Del Castillo Velasco-Herrera & Kosuke Yusa

Authors
  1. Hiroko Koike-Yusa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yilong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E-Pien Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martin Del Castillo Velasco-Herrera
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kosuke Yusa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.Y. conceived the research and wrote the manuscript with comments from all authors. H.K.-Y., E.-P.T. and K.Y. performed the experiments. Y.L. and M.D.C.V.-H. performed the bioinformatics analyses.

Corresponding author

Correspondence to Kosuke Yusa.

Ethics declarations

Competing interests

K.Y. and Y.L. filed a patent application based on the results reported in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–19 and Supplementary Tables 3–7 (PDF 2520 kb)

Supplementary Table 1

Off-target cleavage analyses of the gRNA targeting Site 2 of the Piga gene (with no bulge) (XLSX 101 kb)

Supplementary Table 2

Off-target cleavage analyses of the gRNA targeting Site 2 of the Piga gene (with bulges) (XLSX 157 kb)

Supplementary Table 8

List of shRNAs and oligo seqeunces (XLSX 10 kb)

Supplementary Table 9

List of genes, gRNA target sequences and oligonucleotide sequences used in this study. (XLSX 18 kb)

Supplementary Data 1

(XLSX 35502 kb)

Supplementary Data 2

(XLSX 6296 kb)

Supplementary Data 3

A full list of potential off-target sites (with NGG PAM) of the gRNA targeting site 2 of the Piga gene. (XLSX 338 kb)

Supplementary Data 4

A full list of potential off-target sites (with NAG PAM) of the gRNA targeting Site 2 of the Piga gene. (XLSX 455 kb)

Supplementary Data 5

A full list of potential off-target sites (with bulges) of the gRNA targeting site 2 of the Piga gene. (XLSX 76 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Koike-Yusa, H., Li, Y., Tan, EP. et al. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32, 267–273 (2014). https://doi.org/10.1038/nbt.2800

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2800

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing