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

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.