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.

Predatory feeding behaviour in Pristionchus nematodes is dependent on a phenotypic plasticity and induced by serotonin

“…Furthermore, with recent advancements in methods for generating gene knockouts through CRISPR generated mutations (Lo et al, 2013;Witte et al, 2015), the analysis of genes involved in components of neurotransmitter synthesis and receptor genes can now be investigated. While studies on the dimorphic properties of the P. pacificus mouth form have made great strides in elucidating the intricate developmental and chemical queues driving this process (Bento et al, 2010;Bose et al, 2012;Ragsdale et al, 2013), little was known of the influence of mouth form morphology on behaviour or its evolutionary conservation.…”

Mechanisms of host seeking by parasitic nematodes

“…Mechanistic studies of neural circuit function in parasitic nematodes are now feasible due to the large-scale sequencing of parasitic nematode genomes [91] and the development of new methods for genetic transformation of parasitic worms [92]. In addition, targeted gene disruption has recently been achieved in multiple free-living nematodes using the CRISPR-Cas9 system [93–95], and this system is likely to be applicable to parasitic nematodes. These exciting developments pave the way for in-depth molecular, cellular, and circuit-level analyses of the host-seeking behaviors of parasitic nematodes.…”

Transgenesis inStrongyloidesand related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing

“…The most widely used version of this approach, hereafter referred to as the CRISPR/Cas9 system, involves mutagenesis catalysed by a complex of the Streptococcus pyogenes CRISPR-associated (Cas) endonuclease Cas9 and a small guide RNA (sgRNA) comprising transactivating and sequence-specific targeting domains. The CRISPR/Cas9 system has been adapted for use in non-parasitic nematodes C. elegans (Friedland et al 2013; Lo et al 2013; Waaijers et al 2013), the dioecious Caenorhabditis species 9 (Lo et al 2013) and the necromenic nematode Pristionchus pacificus (Lo et al 2013; Witte et al 2015). Transgenesis in Strongyloides and related genera opens the possibility of using it as a functional genomic tool in these parasitic nematodes.…”