High-efficiency gene editing inAnopheles sinensisusing ReMOT control

Abstract

CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations intoAnopheles sinensis, the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes,AswhiteandAsyellow, using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies inAn. sinensis. Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.

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

“…In this study, we have developed a heritable CRISPR/Cas9 gene-editing system in B. mori using ReMOT control by switching the ovary-targeting ligand from DmP2C to BmOTP. DmP2C was derived from the yolk protein of D. melanogaster and used for the original ReMOT control method, which is effective for gene editing in a various of insect species, including mosquito [12,[16][17][18], N. vitripennis [19], T. castaneum [20], and D. citri [23], as well as I. scapularis [24]. However, the DmP2C ligand failed to target the ovary in the silkworm.…”

“…After injecting the Cas9 ribonucleoprotein complex (Cas9 RNP; DmP2C-Cas9 protein complexed with guide RNAs) into the hemolymph of adult female insects, DmP2C transduces Cas9 RNP directly into the developing ovaries [17]. Using the DmP2C, ReMOT control has been shown to facilitate gene editing in many insect species, including Aedes aegypti [17], Agrotis ipsilon [13], Anopheles stephensi [18], Anopheles sinensis [19], Culex pipiens pallens [20], Nasonia vitripennis [21], Tribolium castaneum [22], Diaphorina citri [23], and even in the Chelicerate, Ixodes scapularis [24]. While the DmP2C ligand did not function robustly in Bemisia tabaci, an ovary-targeting peptide ligand of endogenous vitellogenin protein (BtKV) was identified to effectively mediate gene editing [25].…”