Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus.
Dev Genes Evol
; 225(1): 55-62, 2015 Jan.
Article
em En
| MEDLINE
| ID: mdl-25548084
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas de Inativação de Genes
/
Sistemas CRISPR-Cas
/
Nematoides
Limite:
Animals
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article