CRISPR-Cas9-Mediated Mutagenesis of the Asian Citrus Psyllid, Diaphorina citri

Abstract

The most devastating disease affecting the global citrus industry is Huanglongbing (HLB), caused by the pathogen Candidatus Liberibacter asiaticus. HLB is primarily spread by the insect vector Diaphorina citri (Asian Citrus Psyllid). To counteract the rapid spread of HLB by D. citri, traditional vector control strategies such as insecticide sprays, the release of natural predators, and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To further expand the available tools for D. citri control via generating specific modifications of the D. citri genome, we have developed protocols for CRISPR/Cas9-based genetic modification. Until now, genome editing in D. citri has been challenging due to the general fragility and size of D.citri eggs. Here we present optimized methods for collecting and preparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and alternative methods (ReMOT Control) for injecting RNP into the hemocoel of adult females for ovarian transduction. Using these methods, we have generated visible somatic mutations, indicating their suitability for gene editing in D. citri. These methods represent the first steps towards advancing D. citri research in preparation for future genetic-based systems for controlling HLB. Introduction .

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].…”