BAPC-assistedCRISPR/Cas9 System: Targeted Delivery into Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera)

Abstract

Innovative gene targeting strategies are often limited in application across arthropod species due to problems with successful delivery. In hemipterans, embryonic injections often used to deliver CRISPR components fail due to nearly complete embryo mortality. The Asian citrus psyllid, Diaphorina citri, Kuwayama, (Hemiptera: Liviidae), is the vector for a pathogenic bacterium, Candidatus Liberibacter asiaticus, CLas, which is devastating the U.S. citrus industries. The disease called, Huanglongbing, HLB, (aka. Citrus greening disease), is transmitted during psyllid feeding. Infection causes severe tree decline, loss of fruits, and eventually tree death. The citrus tree pathogen, CLas, is a fastidious alpha-proteobacterium, which has spread into all citrus growing regions worldwide. The economic losses are estimated in the billions of dollars, in U.S.A., Brazil, and China. Innovative technologies aimed at reducing psyllid populations using targeting RNA suppression, like RNAi, or gene-editing tools, like CRISPR/Cas9 have potential to reduce psyllid vectors and the pathogen in a highly specific manner. Breakthroughs that improve gene editing in psyllids, such as the BAPC-assisted-CRISPR/Cas9 System, enabled delivery by injection of CRISPR/Cas9 components directly into nymphs and adult females. Injection near ovaries produced heritable germline gene editing in subsequent generations. This method opens the world of gene editing across arthropods and bypasses the need for microinjection of eggs. Effective development of therapeutic treatments to reduce insect vectors, and stop pathogen transmission would provide sustainable citrus and grapevine industries.

Unravelling the Pathogenesis and Molecular Interactions of Liberibacter Phytopathogens with Their Psyllid Vectors

“…Other novel non-transgenic delivery technologies, including peptide-based delivery vehicles, polymer or liposomic nanoparticles, and viral-like particles, also could be effective RNAi-based mechanisms to disrupt pathogen transmission. Peptide-based nano-assemblies, such as branched amphiphilic peptide capsules (BAPC), which are self-assembling peptide nano-capsular spheres, are also being used as a delivery vehicle for dsRNAs in insects [129,137]. Finally, CRISPR-based gene editing might modulate insect-host response to liberibacter infection.…”

“…Finally, CRISPR-based gene editing might modulate insect-host response to liberibacter infection. Injecting CRISPR/Cas9 components directly into nymphs and adults have shown heritable germline gene editing in subsequent generations in D. citri [137,138].…”

Progress in the use of genetic methods to study insect behavior outside Drosophila

“…In most cases, components of the CRISPR/Cas9 system (Cas9, sgRNAs and a DNA homology template) are injected into a pre-blastoderm embryo. To overcome high costs and workload, and embryo lethality associated with injection, new methods of delivery directly into a gravid female are now being developed [31,32]. Since its introduction as a gene editing tool in 2012, CRISPR/Cas9 system has advanced research in many insect species, including flies [33][34][35][36][37][38], sandflies [39], mosquitoes [40][41][42][43], moths [13,[44][45][46][47], butterflies [48,49], crickets [50], locusts [51], planthoppers [52], honeybees [53,54], wasps [55], ants [11,12], beetles [56], aphids [57] and psyllid bugs [31].…”

Germline mutagenesis ofNasonia vitripennisthrough ovarian delivery of CRISPR-Cas9 ribonucleoprotein

“…Recently, two alternative methods that bypass the requirement for embryonic microinjection have been developed (Chaverra-Rodriguez et al, 2018;Dermauw et al, 2020;Heu et al, 2020;Hunter et al, 2018;Macias et al, 2020). Receptor-Mediated Ovary Transduction of Cargo (ReMOT) involves delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex (Cas9 with a singleguide RNA (sgRNA)) into insect ovaries.…”

“…BAPC can thus facilitate the uptake of DNA and potentially other types of nucleic acid in vitro and in vivo. It has been used for delivery of dsRNA and plasmid DNA in cultured insect and animal cells (Avila et al, 2018;Barros et al, 2016) and recently it has been used to improve the delivery of CRISPR components into adult ovaries to facilitate heritable gene editing in the Asian citrus psyllid Diaphorina citri (Hunter et al, 2018).…”