The Development and Expansion of in vivo Germline Editing Technologies in Arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and Beyond

Abstract

In the past 20 years, sequencing technologies have led to easy access to genomic data from non-model organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene editing vectors into early-stage embryos have been used for arthropod transgenesis and the discovery of CRISPR-Cas technologies allowed for targeted mutagenesis and the creation of knock-outs or knock-ins in arthropods. Receptor-Mediated Ovary transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which requires expensive equipment and extensive hands-on training. ReMOT Control's main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges on the application across species after 5 years since its development, including novel extensions of the technique such as DIPA-CRISPR.

Direct Parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis

“…ReMOT utilizes a modified Cas9 protein fused with a peptide ligand called P2C, which is derived from the D. melanogaster yolk protein precursor ( Chaverra-Rodriguez et al 2018 ). P2C–Cas9 and sgRNAs are combined and co-injected into adult females, and the RNP particles are imported into the oocytes of egg chambers undergoing vitellogenesis ( Terradas et al 2023 ). This approach was found to yield a G0 gene-editing efficiency (GEF: The proportion of edited G0 individuals out of the total number of G0 s) of ∼1.5% in the mosquito Ades aegypti ( Chaverra-Rodriguez et al 2018 ).…”