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Dev Genes Evol. 2015 Jan;225(1):55-62. doi: 10.1007/s00427-014-0486-8. Epub 2014 Dec 30.

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

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

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.

PMID:
25548084
DOI:
10.1007/s00427-014-0486-8
[Indexed for MEDLINE]
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