Biology subjects

Ireri, S. W.

Publications and source records attributed to Ireri, S. W..

3 recordsLinked to original sources

bioRxiv2026

A modular bacterial platform for tunable double-stranded RNA delivery and RNA interference in nematode Caenorhabditis elegans

RNA interference (RNAi), one of the major molecular tools in model nematode Ceanorhabditis elegans, relies on feeding engineered bacteria that express double-stranded RNA (dsRNA), which modulates animal host gene expression in a programmable manner. Currently no synthetic biology toolkit exits in C. elegans RNAi, while dsRNA circuits construction is restricted to one type of architecture. To facilitate systematic strategies for dsRNA circuit design and expression, here, we performed strain-specific screen of synthetic promoters, based on which we developed seventeen modular genetic parts compatible for rapid assembly of dsRNA expression constructs in Escherichia coli HT115(DE3). We validated dsRNA production in vitro and assessed RNAi efficiency in live animals by feeding. As a proof of concept, a constitutive dsRNA circuit achieved rapid and near-complete gene knockdown, whereas a Ptac-driven circuit enabled tunable, partial silencing while minimizing the leakiness commonly observed in standard feeding RNAi systems. Together, this work establishes a synthetic biology toolkit for programmable dsRNA delivery, enabling precise control of RNAi outcomes from partial to complete gene silencing in C. elegans.

bioengineering↗

bioRxiv2025

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. SUMMARYThis article demonstrates CRISPR-Cas9 mediated genome engineering in Steinernema hermaphroditum, an entomopathogenic (EPN: insect-parasitic) nematode and an emerging genetic model. The described technology is useful for creating mutants allowing for the elucidation of gene functions in the nematode biology that is relevant to mutualistic and parasitic symbiosis.

genetics↗

bioRxiv2025

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.

genetics↗