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July 20th, 2025
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Carnegie Institution for Science
genetics
biorxiv

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

Ireri, S. W.Open in Google Scholar•Cao, M.Open in Google Scholar

The entomopathogenic nematodes (EPNs) of the genus Steinernema serve as a valuable experimental model for studying microbial symbiosis and are economically important as organic pest control agents 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.

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