Molecular tool development in traditionally non-tractable animals opens new avenues to study gene functions in the relevant ecological context. Entomopathogenic nematodes (EPN) Steinernema and their symbiotic bacteria of Xenorhabdus spp are a valuable experimental system in the laboratory and are applicable in the field to promote agricultural productivity. The infective juvenile (IJ) stage of the nematode packages mutualistic symbiotic bacteria in the intestinal pocket and invades insects that are agricultural pests. The lack of consistent and heritable genetics tools in EPN targeted mutagenesis severely restricted the study of molecular mechanisms underlying both parasitic and mutualistic interactions. Here, I report a protocol for CRISPR-Cas9 based genome-editing that is successful in two EPN species, S. carpocapsae and S. hermaphroditum . I adapted a gonadal microinjection technique in S. carpocapsae , which created on-target modifications of a homologue Sc-dpy-10 (cuticular collagen) by homology-directed repair. A similar delivery approach was used to introduce various alleles in S. hermaphroditum including Sh-dpy-10 and Sh-unc-22 (a muscle gene), resulting in visible and heritable phenotypes of dumpy and twitching, respectively. Using conditionally dominant alleles of Sh-unc-22 as a co-CRISPR marker, I successfully modified a second locus encoding Sh-Daf-22 (a homologue of human sterol carrier protein SCPx), predicted to function as a core enzyme in the biosynthesis of nematode pheromone that is required for IJ development. As a proof of concept, Sh-daf-22 null mutant showed IJ developmental defects in vivo ( in insecta) . This research demonstrates that Steinernema spp are highly tractable for targeted mutagenesis and has great potential in the study of gene functions under controlled laboratory conditions within the relevant context of its ecological niche.