The ABC's of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera, and Ceratitis

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2017
Publication Date: 10/6/2017
Citation: Sim, S.B., Kauwe, A.N., Ruano, R.E., Rendon, P., Geib, S.M. 2017. The ABC's of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera, and Ceratitis. Insect Molecular Biology. 28(2):277-289. https://doi.org/10.1111/imb.12550.
DOI: https://doi.org/10.1111/imb.12550

Interpretive Summary: The development of agents for genetic pest management of fruit flies can be achieved by radiation, chemical mutagenesis, and transgenic techniques. The CRISPR/Cas9 system is a new method for inducing site-specific mutations has recently emerged as a powerful tool that should work universally in all prokaryotic and eukaryotic species. However, to apply this technology to the control of tephritid fruit fly pests, it is necessary to develop and demonstrate methods for inducing germline mutations using CRISPR/Cas9 in every descructive species. In this manuscript, we describe methods we have developed for three species of Tephritidae in the genera Anastrepha, Bactrocera, and Ceratitis and report the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. The methods in this manuscript can serve as a resource for others attempting to develop CRISPR/Cas9 methods in their system and a guide for others who require a mobile microinjection station.

Technical Abstract: Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by non-homologous end join repair of CRISPR/Cas9 nuclease has been demonstrated to be an efficient method for creating knock-out mutants and can be utilized to create germline mutations in Tephritidae. In this manuscript, we describe detailed methods to knockout the white gene in three species in the genera Anastrepha, Bactrocera, and Ceratitis, and we report the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the phenotypic variables in tephritid systems that directed our method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.