Cas9-Mediated Gene Editing Using Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control in Bombyx mori

Abstract

Lepidoptera is one of the most speciose insect orders, causing enormous damage to agricultural and forest crops. Although genome editing has been achieved in a few Lepidoptera for insect controls, most techniques are still limited. Here, by injecting female pupae of the Lepidoptera model species, Bombyx mori, gene editing was established using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control technique. We identified a B. mori oocytes-targeting peptide ligand (BmOTP, a 29 aa of vitellogenin N-terminal of silkworms) with a highly conserved sequence in lepidopteran insects that could efficiently deliver mCherry into oocytes. When BmOTP was fused to CRISPR-associated protein 9 (Cas9) and the BmOTP-Cas9 ribonucleoprotein complex was injected into female pupae, heritable editing of the offspring was achieved in the silkworms. Compared with embryo microinjection, individual injection is more convenient and eliminates the challenge of injecting extremely small embryos. Our results will significantly facilitate the genetic manipulation of other lepidopteran insects, which is essential for advancing lepidopteran pest control.