CRISPR-Cas9 based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines

Entomopathogenic nematodes (EPNs) from the genus Steinernema and Heterorhabditis form mutualistic relationships with symbiotic bacteria from the genus Xenorhabdus and Photorhabdus , respectively. Together, these nematode-bacterium pairs infect and kill insect hosts—primarily larvae from the orders Lepidoptera and Coleoptera . This tripartite interaction provides a powerful model for investigating the molecular mechanisms underlying mutualism and parasitism. A key step toward this goal is the development of a genetically tractable EPN. While RNAi has been applied in some EPN species, stable, transgenerational genetic tools remain limited. Here, we establish a robust CRISPR-Cas9 system in the emerging model Steinernema hermaphroditum , a species that is easily cultivated in both in vivo and in vitro conditions and amenable to gonadal microinjection. Notably, its hermaphroditic reproduction simplifies the generation of genetically stable mutant lines. We present a detailed protocol for efficient, targeted gene knockout via microinjection in S. hermaphroditum . As a proof-of-concept, we knocked out a conserved homologue, unc-22 , which causes a twitching phenotype. The CRISPR-Cas9 based genome editing in S. hermaphroditum has potential to be used to express transgene, or to be adapted to other EPN species that are applicable to benefit agriculture.