CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa

Authors: LI, JIANWEI - UNIVERSITY OF FLORIDA; Handler, Alfred - Al

Submitted to: Gene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2018
Publication Date: 1/3/2019
Citation: Li, J., Handler, A.M. 2019. CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa. Gene. 691:160-166. https://doi.org/10.1016/j.gene.2018.12.055.
DOI: https://doi.org/10.1016/j.gene.2018.12.055

Interpretive Summary: The creation of genetically modified strains for the development of more effective biological control programs for insects of agricultural and medical importance is a major goal of the USDA, Agriculture Research Service, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. A major roadblock to the efficient creation of these strains, and the design of novel strategies, is the random insertion of DNA into the genome using current gene-transfer vector systems, and the occasional need to eliminate function of some active genes in the host strain. Both of these drawbacks can be eliminated by the successful use of new gene-editing techniques based on the CRISPR/Cas9 system. In this report, we describe experiments that test the CRISPR/Cas9 system for the first time in the Caribbean fruit fly, Anastrepha suspensa, by eliminating the function of a previous genetic modification (an EGFP fluorescent protein marker gene) and an existing gene, known as transformer-2, critical to sex-specific development and fertility. The function of both genes was eliminated specifically and with high efficiency, indicating that CRISPR/Cas9 can be used in the Caribfly, and related pest species, for new genetic modifications that improve the control of their population size in the field.

Technical Abstract: CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, having deletions ranging from 2 to 5 nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Mutations were identified phenotypically in G0 adult flies based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA are known not to have a phenotypic effect in XY males. Consistent with this expectation, 12 types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5’ sgRNA-1 target site in intersexual adults at a frequency of 81%. However, the 3’ sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.