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.

Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

This article has been updated

Abstract

Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Adaptation of the CRISPR-Cas9 system for C. elegans. (a) Schematic of the Cas9 nuclease and sgRNA.
Figure 2: Efficiency of Cas9-triggered homologous recombination in C. elegans.
Figure 3: Tagging of endogenous nmy-2 with gfp.
Figure 4: Tagging of endogenous his-72 with gfp.
Figure 5: Targeted mutations in an endogenous gene.

Change history

  • 16 September 2013

    In the version of this article initially published online, the phrase "insertion of gfp into the nmy-2 gene and a 23-bp loxP site" should have read "insertion of gfp into the nmy-2 gene and a 34-bp loxP site." In the Online Methods, under the heading "Single-copy transgene insertion with MosSCI," "pGH8 (Prab-8::mCherry neuronal co-injection marker)" should have read "pGH8 (Prab-3::mCherry neuronal co-injection marker)." The errors have been corrected for the print, PDF and HTML versions of this article.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    CAS  Article  Google Scholar 

  3. 3

    DiCarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    CAS  Article  Google Scholar 

  4. 4

    Gratz, S.J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194, 1029–1035 (2013).

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    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).

    CAS  Article  Google Scholar 

  7. 7

    Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Friedland, A.E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741–743 (2013).

    CAS  Article  Google Scholar 

  9. 9

    Bassett, A.R., Tibbit, C., Ponting, C.P. & Liu, J.-L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 4, 220–228 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Chang, N. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465–472 (2013).

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    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).

    CAS  Article  Google Scholar 

  13. 13

    Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    CAS  Article  Google Scholar 

  15. 15

    Lo, T.-W. et al. Heritable genome editing using TALENs and CRISPR/Cas9 to engineer precise insertions and deletions in evolutionarily diverse nematode species. Genetics doi:10.1534/genetics.113.155382 (9 August 2013).

  16. 16

    Gerstein, M.B. et al. Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project. Science 330, 1775–1787 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Frøkjær-Jensen, C., Davis, M.W., Ailion, M. & Jorgensen, E.M. Improved Mos1-mediated transgenesis in C. elegans. Nat. Methods 9, 117–118 (2012).

    Article  Google Scholar 

  18. 18

    Robert, V. & Bessereau, J.-L. Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks. EMBO J. 26, 170–183 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Robert, V.J., Davis, M.W., Jorgensen, E.M. & Bessereau, J.-L. Gene conversion and end-joining-repair double-strand breaks in the Caenorhabditis elegans germline. Genetics 180, 673–679 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Frøkjær-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

    Article  Google Scholar 

  21. 21

    Frøkjær-Jensen, C. et al. Targeted gene deletions in C. elegans using transposon excision. Nat. Methods 7, 451–453 (2010).

    Article  Google Scholar 

  22. 22

    Vallin, E. et al. A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community. PLoS ONE 7, e30482 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    CAS  Article  Google Scholar 

  24. 24

    Kelly, W.G., Xu, S., Montgomery, M.K. & Fire, A. Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene. Genetics 146, 227–238 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Sarov, M. et al. A genome-scale resource for in vivo tag-based protein function exploration in C. elegans. Cell 150, 855–866 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Nance, J., Munro, E.M. & Priess, J.R. C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation. Development 130, 5339–5350 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Guo, S. & Kemphues, K.J. A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 382, 455–458 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Berezikov, E., Bargmann, C.I. & Plasterk, R.H.A. Homologous gene targeting in Caenorhabditis elegans by biolistic transformation. Nucleic Acids Res. 32, e40 (2004).

    Article  Google Scholar 

  30. 30

    Ferguson, E.L. & Horvitz, H.R. Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenorhabditis elegans. Genetics 110, 17–72 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Tan, P.B., Lackner, M.R. & Kim, S.K. MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell 93, 569–580 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. doi:10.1038/nbt.2623 (23 June 2013).

  33. 33

    Granger, L., Martin, E. & Ségalat, L. Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study. Nucleic Acids Res. 32, e117 (2004).

    Article  Google Scholar 

  34. 34

    Williams, D.C., Boulin, T., Ruaud, A.-F., Jorgensen, E.M. & Bessereau, J.-L. Characterization of Mos1-mediated mutagenesis in Caenorhabditis elegans: a method for the rapid identification of mutated genes. Genetics 169, 1779–1785 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Shirayama, M. et al. piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell 150, 65–77 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Lam, A.J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Stiernagle, T. in WormBook (ed. The C. elegans Research Community) doi:10.1895/wormbook.1.101.1 (2006).

  40. 40

    Redemann, S. et al. Codon adaptation-based control of protein expression in C. elegans. Nat. Methods 8, 250–252 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Macosko, E.Z. et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171–1175 (2009).

    CAS  Article  Google Scholar 

  42. 42

    Merritt, C., Rasoloson, D., Ko, D. & Seydoux, G. 3′ UTRs are the primary regulators of gene expression in the C. elegans germline. Curr. Biol. 18, 1476–1482 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Maduro, M. & Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141, 977–988 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Zhang, Y., Chen, D., Smith, M.A., Zhang, B. & Pan, X. Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity. PLoS ONE 7, e31849 (2012).

    CAS  Article  Google Scholar 

  45. 45

    Hoogewijs, D., Houthoofd, K., Matthijssens, F., Vandesompele, J. & Vanfleteren, J.R. Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol. Biol. 9, 9 (2008).

    Article  Google Scholar 

  46. 46

    Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank C. Frøkjær-Jensen (University of Utah) for sharing strains, plasmids and protocols; K. Kemphues (Cornell University) for antibodies; and K. Bloom, A. Maddox, G. Monsalve, N. Pujol, K. Slep, S. Taubert, K. Yamamoto and members of the Goldstein lab for helpful suggestions and comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the US National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). This work was supported by NIH T32 CA009156 and a Howard Hughes postdoctoral fellowship from the Helen Hay Whitney Foundation (D.J.D.); a postdoctoral fellowship from the Canadian Institutes of Health Research (award #234765) (J.D.W.); NIH R01 GM085309 (D.J.R.); NIH CA20535 and US National Science Foundation (NSF) MCB 1157767 (K. Yamamoto); and NIH R01 GM083071 and NSF IOS 0917726 (B.G.).

Author information

Affiliations

Authors

Contributions

D.J.D. and J.D.W. jointly conceived of the project, and all authors discussed and contributed to the experimental design. D.J.D. performed the experiments. D.J.D. and D.J.R. analyzed the data. D.J.D. prepared the manuscript, and all authors discussed and contributed to the final version.

Corresponding author

Correspondence to Daniel J Dickinson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Tables 1–6 and Supplementary Protocol (PDF 4030 kb)

NMY-2–GFP dynamics in zuIs45 and knock-in embryos

1-cell embryos homozygous for either zuIs45 or the nmy-2::gfp knock-in were placed side by side on the same coverslip and filmed simultaneously. Still images from this movie are shown in Fig. 3e. (AVI 53807 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dickinson, D., Ward, J., Reiner, D. et al. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods 10, 1028–1034 (2013). https://doi.org/10.1038/nmeth.2641

Download citation

Further reading

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