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

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.

Keywords

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)

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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

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