BAPC-assisted CRISPR/Cas9 delivery system heritable germline gene editing with adult and nymph injections (Arthropoda: Hemiptera: Psyllids, Leafhoppers, Whitefly)

Authors: Hunter, Wayne; TOMICH, JOHN - Kansas State University

Submitted to: International Research Conference on Huanglongbing
Publication Type: Proceedings
Publication Acceptance Date: 2/18/2019
Publication Date: 3/14/2019
Citation: Hunter, W.B., Tomich, J. 2019. BAPC-assisted CRISPR/Cas9 delivery system heritable germline gene editing with adult and nymph injections (Arthropoda: Hemiptera: psyllids, leafhoppers, whitefly). 6th International Research Conference on Huanglongbing. March 10-15, Riverside, California. 6, p. 324.

Interpretive Summary: Citrus insect pests, like Asian citrus psyllid transmit Liberibacter bacterial pathogens during feeding. A new method represents a major breakthrough in management of insect vectors. Using gene targeting permits safer, more specific, management methods to reduce insect pests and pathogens. The new method, called "BAPC-assisted-Delivery", (Branched Amphiphilic Peptide Capsules, BAPC™, Phoreus™ Biotechnology, Inc) improved gene knockouts in adult Asian Citrus Psyllid, Diaphorina citri. The method permits researchers to rapidly test potential gene targets. Using the method evaluations identified a suitable gene that may be used in reducing psyllid populations, and thereby reduce the spread of Huanglongbing.

Technical Abstract: CA new method called BAPC-assisted-CRISPR-Delivery System, for heritable gene editing by treating nymphs and adult Insects, Hemiptera, was demonstrated. The Branched Amphiphilic Peptide Capsules, BAPC™, (Phoreus™ Biotechnology, Inc) improves delivery of CRISPR components (Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein, Cas9), plasmids, and ribonucleic acids, Ribonucleic acid (RNA), to increase heritable gene editing and gene targeting, or expression, in insect nymphs and adults (Asian Citrus Psyllid- Diaphorina citri ). The method bypasses traditional microinjection of eggs. This reports the first heritable gene knock-outs, KO, using BAPC-assisted-CRISPR-Cas9, produced second generation mutants from injected adult psyllid females. The knock out (KO) target was the thioredoxin gene, resulting in 550 nucleotides deletion in genomic DNA of target insects. In psyllids the thioredoxin gene, TXT, and Vermillion, Vm, knock outs produced changes in physiology and eye color, respectively. Strategies to alter the psyllid vector into a non-vector of the bacterium, Las. One function of TXT is to promote development. When knocked out development is slower, reduced survival, and reduced adult lifespan and fecundity. Reduced fecundity and slow development traits would reduce insect populations by increasing exposure time to parasitism and predation with more time, extra 2 to 3 weeks longer to attack nymphal stages. Emerging adults only lived one-third as long. Classical CRISPR/Cas9 injections of nymphs and pupae produced Vm-KO, white eye phenotypes. The BAPC™-assisted delivery system advances gene editing across all hemipterans and insects by permitting the use of nymphs and adults. The technology opens strategies to protect citrus and food crops from insect vectors and pathogens [citrusgreening.org]. Funding in part: USDA-NIFA 2014-70016-23028. Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease.