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Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus

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Abstract

The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various –omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes.

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References

  • Borchert N, Dieterich C, Krug K, Schutz W, Jung S, Nordheim A, Sommer RJ, Macek B (2010) Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. Genome Res 20:837–846

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW (2013) Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195:1167–1171

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cho SW, Lee J, Carroll D, Kim JS (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–1180

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cinkornpumin JK, Hong RL (2011) RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic nematode Pristionchus pacificus. J Vis Exp e3270

  • Dieterich C, Clifton SW, Schuster LN, Chinwalla A, Delehaunty K, Dinkelacker I, Fulton L, Fulton R, Godfrey J, Minx P, Mitreva M, Roeseler W, Tian H, Witte H, Yang SP, Wilson RK, Sommer RJ (2008) The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism. Nat Genet 40:1193–1198

    Article  CAS  PubMed  Google Scholar 

  • Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  CAS  PubMed  Google Scholar 

  • Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10:741–743

    Article  CAS  PubMed  Google Scholar 

  • Herrmann M, Kienle S, Rochat J, Mayer WE, Sommer RJ (2010) Haplotype diversity of the nematode Pristionchus pacificus on Réunion in the Indian Ocean suggests multiple independent invasions. Biol J Linn Soc 100:170–179

    Article  Google Scholar 

  • Irion U, Krauss J, Nüsslein-Volhard C (2014) Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development in press

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  PubMed  Google Scholar 

  • Jungblut B, Sommer RJ (1998) The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates. Curr Biol 8:775–778

    Article  CAS  PubMed  Google Scholar 

  • Katic I, Grosshans H (2013) Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics 195:1173–1176

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kenning C, Kipping I, Sommer RJ (2004) Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus. Genesis 40:176–183

    Article  CAS  PubMed  Google Scholar 

  • Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nature Reviews Genetics 15:321–334

    Article  CAS  PubMed  Google Scholar 

  • Kim H, Ishidate T, Ghanta KS, Seth M, Conte D Jr, Shirayama M, Mello CC (2014) A Co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197:1069–1080

    Article  PubMed Central  PubMed  Google Scholar 

  • Lee JS, Kwak SJ, Kim J, Kim AK, Noh HM, Kim JS, Yu K (2014) RNA-guided genome editing in Drosophila with the purified Cas9 protein. G3 4, 1291–1295

  • Lo TW, Pickle CS, Lin S, Ralston EJ, Gurling M, Schartner CM, Bian Q, Doudna JA, Meyer BJ (2013) Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195:331–348

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Morgan K, McGaughran A, Villate L, Herrmann M, Witte H, Bartelmes G, Rochat J, Sommer RJ (2012) Multi locus analysis of Pristionchus pacificus on La Reunion Island reveals an evolutionary history shaped by multiple introductions, constrained dispersal events and rare out-crossing. Mol Ecol 21:250–266

    Article  PubMed  Google Scholar 

  • Poland JA, Brown PJ, Sorrells ME, Jannink JL (2012) Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS One 7:e32253

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rodelsperger C, Sommer RJ (2011) Computational archaeology of the Pristionchus pacificus genome reveals evidence of horizontal gene transfers from insects. BMC Evol Biol 11:239

    Article  PubMed Central  PubMed  Google Scholar 

  • Rodelsperger C, Neher RA, Weller AM, Eberhardt G, Witte H, Mayer WE, Dieterich C, Sommer RJ (2014) Characterization of genetic diversity in the nematode Pristionchus pacificus from population-scale resequencing data. Genetics 196:1153–1165

    Article  PubMed  Google Scholar 

  • Schlager B, Wang X, Braach G, Sommer RJ (2009) Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus. Genesis 47:300–304

    Article  CAS  PubMed  Google Scholar 

  • Sommer RJ (2009) The future of evo-devo: model systems and evolutionary theory. Nat Rev Genet 10:416–422

    CAS  PubMed  Google Scholar 

  • Sommer RJ, McGaughran A (2013) The nematode Pristionchus pacificus as a model system for integrative studies in evolutionary biology. Mol Ecol 22:2380–2393

    Article  PubMed  Google Scholar 

  • Sommer RJ, Sternberg PW (1996) Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 6:52–59

    Article  CAS  PubMed  Google Scholar 

  • Srinivasan J, Sinz W, Jesse T, Wiggers-Perebolte L, Jansen K, Buntjer J, van der Meulen M, Sommer RJ (2003) An integrated physical and genetic map of the nematode Pristionchus pacificus. Mol Genet Genomics 269:715–722

    Article  CAS  PubMed  Google Scholar 

  • Stein LD, Bao Z, Blasiar D, Blumenthal T, Brent MR, Chen N, Chinwalla A, Clarke L, Clee C, Coghlan A, Coulson A, D’Eustachio P, Fitch DH, Fulton LA, Fulton RE, Griffiths-Jones S, Harris TW, Hillier LW, Kamath R, Kuwabara PE, Mardis ER, Marra MA, Miner TL, Minx P, Mullikin JC, Plumb RW, Rogers J, Schein JE, Sohrmann M, Spieth J, Stajich JE, Wei C, Willey D, Wilson RK, Durbin R, Waterston RH (2003) The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics. PLoS Biol 1:E45

    Article  PubMed Central  PubMed  Google Scholar 

  • Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stiernagle T (1999) Maintenance of C. elegans. In: Hope IA (ed) C. elegans a practical approach. Oxford University Press, Oxford, pp 51–67

    Google Scholar 

  • Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS (2014) Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24:125–131

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiacovo MP (2013) Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics 195:1181–1185

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Waaijers S, Boxem M (2014) Engineering the Caenorhabditis elegans genome with CRISPR/Cas9. Methods 68:381–388

    Article  CAS  PubMed  Google Scholar 

  • Waaijers S, Portegijs V, Kerver J, Lemmens BB, Tijsterman M, van den Heuvel S, Boxem M (2013) CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans. Genetics 195:1187–1191

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Meyer BJ (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333:307

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

We thank James Lightfoot for the language assistance and Seokjoong Kim (ToolGen Inc.) for the valuable technical suggestions. This work was funded by the Max Planck Society. J.-S.K. was supported by a grant from IBS (IBS-R021-D1).

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Authors and Affiliations

  1. Department Evolutionary Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076, Tübingen, Germany

    Hanh Witte, Eduardo Moreno, Christian Rödelsperger, Adrian Streit & Ralf J. Sommer

  2. Department of Chemistry, Seoul National University, Seoul, 151-747, South Korea

    Jungeun Kim & Jin-Soo Kim

  3. Center for Genome Engineering, Institute for Basic Science, Seoul, 151-747, South Korea

    Jin-Soo Kim

Authors
  1. Hanh Witte
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  2. Eduardo Moreno
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  3. Christian Rödelsperger
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  4. Jungeun Kim
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  5. Jin-Soo Kim
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  6. Adrian Streit
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  7. Ralf J. Sommer
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Corresponding author

Correspondence to Ralf J. Sommer.

Additional information

Communicated by Volker G. Hartenstein

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Suppl. Fig. 1

Maximum likelihood tree including dpy-1 homologs from C. elegans (CEL), C. briggsae (CBR), P. pacificus (PPA) and P. exspectatus (PEX). For most genes, the topology of the subtree exactly matches the species phylogeny of the four nematodes, suggesting that one-to-one orthology exists for most members in this gene family. (PDF 30 kb)

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Witte, H., Moreno, E., Rödelsperger, C. et al. Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus . Dev Genes Evol 225, 55–62 (2015). https://doi.org/10.1007/s00427-014-0486-8

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  • DOI: https://doi.org/10.1007/s00427-014-0486-8

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