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

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Witte,  H
Department Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Max Planck Society;

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Moreno,  E
Department Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Max Planck Society;

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Rödelsperger,  C
Department Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Max Planck Society;

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Streit,  A
Department Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Max Planck Society;

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Sommer,  RJ
Department Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Max Planck Society;

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Citation

Witte, H., Moreno, E., Rödelsperger, C., Kim, J., Kim, J.-S., Streit, A., et al. (2015). Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus. Development Genes and Evolution, 225(1), 55-62. doi:10.1007/s00427-014-0486-8.


Cite as: http://hdl.handle.net/21.11116/0000-000A-A4C4-F
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.