Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing

TLDR

Cas9-mediated gene editing is powerful for arthropods, yet current embryonic microinjection approaches are challenging, species‑limited, and inefficient, prompting the search for taxa‑specific ovary‑ligand pairs to broaden ReMOT Control. The authors develop ReMOT Control to deliver Cas9 RNP into the germline of adult female mosquitoes. ReMOT Control injects Cas9 RNP into adult females, using a peptide‑mediated transduction pathway to transport the complex from hemolymph into oocytes. A P2C peptide enables heritable editing with up to 0.3 mutants per mosquito and functions across six mosquito species.

Abstract

Abstract Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand–receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.