Development Genes and Evolution

, Volume 225, Issue 1, pp 55–62 | Cite as

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

  • Hanh Witte
  • Eduardo Moreno
  • Christian Rödelsperger
  • Jungeun Kim
  • Jin-Soo Kim
  • Adrian Streit
  • Ralf J. Sommer
Technical Note


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.


Pristionchus pacificus Nematodes CRISPR/Cas9 Gene knockout 

Supplementary material

427_2014_486_MOESM1_ESM.pdf (31 kb)
Suppl. Fig. 1Maximum 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)


  1. 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 20:837–846PubMedCentralPubMedCrossRefGoogle Scholar
  2. Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW (2013) Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195:1167–1171PubMedCentralPubMedCrossRefGoogle Scholar
  3. Cho SW, Lee J, Carroll D, Kim JS (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–1180PubMedCentralPubMedCrossRefGoogle Scholar
  4. Cinkornpumin JK, Hong RL (2011) RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic nematode Pristionchus pacificus. J Vis Exp e3270Google Scholar
  5. 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 40:1193–1198PubMedCrossRefGoogle Scholar
  6. 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 391:806–811PubMedCrossRefGoogle Scholar
  7. 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 10:741–743PubMedCrossRefGoogle Scholar
  8. 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 100:170–179CrossRefGoogle Scholar
  9. Irion U, Krauss J, Nüsslein-Volhard C (2014) Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development in press Google Scholar
  10. 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 337:816–821PubMedCrossRefGoogle Scholar
  11. Jungblut B, Sommer RJ (1998) The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates. Curr Biol 8:775–778PubMedCrossRefGoogle Scholar
  12. Katic I, Grosshans H (2013) Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics 195:1173–1176PubMedCentralPubMedCrossRefGoogle Scholar
  13. Kenning C, Kipping I, Sommer RJ (2004) Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus. Genesis 40:176–183PubMedCrossRefGoogle Scholar
  14. Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nature Reviews Genetics 15:321–334PubMedCrossRefGoogle Scholar
  15. 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 197:1069–1080PubMedCentralPubMedCrossRefGoogle Scholar
  16. 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, 1291–1295Google Scholar
  17. 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 195:331–348PubMedCentralPubMedCrossRefGoogle Scholar
  18. 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 21:250–266PubMedCrossRefGoogle Scholar
  19. 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:e32253PubMedCentralPubMedCrossRefGoogle Scholar
  20. Rodelsperger C, Sommer RJ (2011) Computational archaeology of the Pristionchus pacificus genome reveals evidence of horizontal gene transfers from insects. BMC Evol Biol 11:239PubMedCentralPubMedCrossRefGoogle Scholar
  21. 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 196:1153–1165PubMedCrossRefGoogle Scholar
  22. 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 47:300–304PubMedCrossRefGoogle Scholar
  23. Sommer RJ (2009) The future of evo-devo: model systems and evolutionary theory. Nat Rev Genet 10:416–422PubMedGoogle Scholar
  24. Sommer RJ, McGaughran A (2013) The nematode Pristionchus pacificus as a model system for integrative studies in evolutionary biology. Mol Ecol 22:2380–2393PubMedCrossRefGoogle Scholar
  25. 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 6:52–59PubMedCrossRefGoogle Scholar
  26. 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 269:715–722PubMedCrossRefGoogle Scholar
  27. 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:E45PubMedCentralPubMedCrossRefGoogle Scholar
  28. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67PubMedCentralPubMedCrossRefGoogle Scholar
  29. Stiernagle T (1999) Maintenance of C. elegans. In: Hope IA (ed) C. elegans a practical approach. Oxford University Press, Oxford, pp 51–67Google Scholar
  30. 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 24:125–131PubMedCentralPubMedCrossRefGoogle Scholar
  31. 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 195:1181–1185PubMedCentralPubMedCrossRefGoogle Scholar
  32. Waaijers S, Boxem M (2014) Engineering the Caenorhabditis elegans genome with CRISPR/Cas9. Methods 68:381–388PubMedCrossRefGoogle Scholar
  33. 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 195:1187–1191PubMedCentralPubMedCrossRefGoogle Scholar
  34. 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 333:307PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hanh Witte
    • 1
  • Eduardo Moreno
    • 1
  • Christian Rödelsperger
    • 1
  • Jungeun Kim
    • 2
  • Jin-Soo Kim
    • 2
    • 3
  • Adrian Streit
    • 1
  • Ralf J. Sommer
    • 1
  1. 1.Department Evolutionary BiologyMax Planck Institute for Developmental BiologyTübingenGermany
  2. 2.Department of ChemistrySeoul National UniversitySeoulSouth Korea
  3. 3.Center for Genome EngineeringInstitute for Basic ScienceSeoulSouth Korea

Personalised recommendations