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

• DOI: 10.1007/s00427-014-0486-8
• Publication Date: 2014-12-29T04:39:26Z
• Authors: Hanh Witte; Eduardo Moreno; Christian Rödelsp...; Jungeun Kim; Jin-Soo Kim; Adrian Streit; Ralf J. Sommer
• Keywords: 570; Gene Knockout Techniques; 0303 health sciences; 03 medical and health sciences; Nematoda; 610; Animals; CRISPR-Cas Systems; Cloning, Molecular

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.