title: Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus.
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.
authors.affiliation: Department Evolutionary Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076, Tübingen, Germany.
authors.forename: Hanh
authors.initials: H
authors.lastname: Witte
authors.forename: Eduardo
authors.initials: E
authors.lastname: Moreno
authors.forename: Christian
authors.initials: C
authors.lastname: Rödelsperger
authors.forename: Jungeun
authors.initials: J
authors.lastname: Kim
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.forename: Adrian
authors.initials: A
authors.lastname: Streit
authors.forename: Ralf J
authors.initials: RJ
authors.lastname: Sommer
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D055786:Gene Knockout Techniques; D009348:Nematoda / Q000145:classification / Q000235:genetics*