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Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

Nature Methods volume 10, pages 1028–1034 (2013)Cite this article

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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.

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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.

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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.

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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.).

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Author notes

  1. David J Reiner

    Present address: Present address: Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, Texas, USA.,

Authors and Affiliations

  1. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Daniel J Dickinson & Bob Goldstein

  2. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Daniel J Dickinson, David J Reiner & Bob Goldstein

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    Jordan D Ward

  4. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    David J Reiner

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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.

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Correspondence to Daniel J Dickinson.

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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)

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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

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