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

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.

Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

“…stephensi by ReMOT Control, we utilized the ovary targeting P2C peptide derived from Drosophila melanogaster yolk protein 1 (DmYP1), which we recently reported in Ae. aegypti to mediate delivery of Cas9 RNP into vitellogenic oocytes 8 . However, we found that the Aedes injection parameters were not directly translatable to An.…”

“…In previous work, we used chloroquine to facilitate exit of the Cas9 RNP from the endosome after uptake, but we noted that saponin (Quallaja bark extract) also could be used for this purpose (8). For…”

“…ECFP+, DsRed+ individuals, which are parental in phenotype, were retained from each injection group, separated by sex and then out-crossed en masse to wild-type mosquitoes to test for the presence of that many reagents that mediate endosomal escape are usable for ReMOT control, which will further the adaptability of this technique. The ligand P2C is broadly effective in mosquitoes 8 , and may be useful in other insect species as well. In organisms where P2C is not effective in delivery, other ligands may be identified from proteins intrinsic to that species that are specifically targeted to the germline.…”

“…While many techniques based on CRISPR/Cas9 have improved studies in mosquito genetics, these techniques rely on embryo injection of the Cas9 ribonucleoproten complex (RNP), which requires specialized and expensive equipment and training. To get around this bottleneck, a strategy termed ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) was developed (8) for delivery of gene-editing moieties to the arthropod germline from the hemocoel, allowing targeted and heritable mutations to be made by adult injection instead of embryo microinjection. Proof-of-principle experiments validating ReMOT Control were conducted in Aedes aegypti, but extension of the method to species more recalcitrant to editing (such as Anopheles mosquitoes) was not established 8 .…”

Delivery of genome editing tools by bacterial extracellular vesicles

“…Cas9) in complex with the gRNA, the socalled CRISPR-Cas9 RNA-protein complex (or ribonucleoprotein complex; RNP), which after uptake allows precise genome editing. Currently, efforts are made to deliver RNP complexes via virus-like particles, receptormediated endocytosis or osmocytosis (D'Astolfo et al, 2015;Liu et al, 2015;Yin et al, 2016;Chaverra-Rodriguez et al, 2018).…”