CRISPR-Cas9 gene editing in the agriculturally beneficial entomopathogenic nematode Steinernema feltiae

The entomopathogenic nematodes (EPN) of the genus Steinernema serves as a valuable experimental model for studying microbial symbiosis and is economically important as organic pest control agent in agriculture. Although most Steinernema species are dioecious (male-female), consistent genetic manipulation has thus far only been demonstrated in the hermaphroditic species Steinernema hermaphroditum. In this study, we adapted a CRISPR-Cas9-based gene editing approach to Steinernema feltiae, a dioecious species widely used in agricultural applications. Using gonadal microinjection, we targeted the conserved gene unc-22 in S. feltiae and generated stable mutant lines with large on-target deletions. Mating tests revealed that Sf-unc-22 is X-linked and exhibits a conditionally dominant twitching phenotype. Additionally, Sf-unc-22 mutants display distinct body morphology compared to wild-type nematodes. Homozygous mutant lines can be reliably maintained through cryopreservation. Altogether, our work provides a proof of concept that genetic tools developed in S. hermaphroditum can be effectively adapted to other agriculturally relevant and dioecious Steinernema species-- broadening the scope of molecular genetic research in microbial ecology and enhancing their potential applications in agriculture.