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

• Published in: Nature communications Vol. 9; no. 1; pp. 3008 - 11
• Main Authors: Chaverra-Rodriguez, Duverney, Macias, Vanessa M., Hughes, Grant L., Pujhari, Sujit, Suzuki, Yasutsugu, Peterson, David R., Kim, Donghun, McKeand, Sage, Rasgon, Jason L.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.08.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 14/32; 38/44; 42/35; 42/41; 45/41; 631/1647/1511; 631/208/4041/3196; 82/83; Alleles; Animals; Aquatic insects; Arthropods; Base Sequence; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Crosses, Genetic; Culicidae - embryology; Culicidae - genetics; Drosophila melanogaster - genetics; Embryo, Nonmammalian - metabolism; Embryos; Female; Gene Editing; Genetic modification; Genome editing; Germ Cells - metabolism; Green Fluorescent Proteins - metabolism; Hemolymph; Humanities and Social Sciences; Inheritance Patterns - genetics; Injections; Male; Microinjection; Mosquitoes; multidisciplinary; Mutation - genetics; Oocytes; Oocytes - metabolism; Ovaries; Ovary - metabolism; Ribonucleoproteins - metabolism; Science; Science (multidisciplinary); Sequence Deletion; Species
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-05425-9

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. Delivery of Cas9 ribonucleoprotein complexes can be done via microinjection into eggs, though this is a technically challenging procedure. Here the authors demonstrate ReMOT Control, delivery of RNPs to the mosquito germline by tagging with peptide derived from yolk protein 1.