CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in fall armyworm moth (Spodoptera frugiperda)

Authors: WU, KE - University Of Florida; Shirk, Paul; TAYLOR, CAITLIN - University Of Florida; Furlong, Richard; Shirk, Bryce; PINHEIRO, DANIELE - University Of Florida; SIEGFRIED, BLAIR - University Of Florida

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2018
Publication Date: 12/6/2018
Citation: Wu, K., Shirk, P.D., Taylor, C.E., Furlong, R.B., Shirk, B.D., Pinheiro, D.H., Siegfried, B.D. 2018. CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in fall armyworm moth (Spodoptera frugiperda). PLoS One. 13(2):e0208647. https://doi.org/10.1371/journal.pone.0208647.
DOI: https://doi.org/10.1371/journal.pone.0208647

Interpretive Summary: The fall armyworm (FAW) is an important pest of maize in the Americas and has recently been introduced into Africa. As with most major pests, the fall armyworm populations in the field have developed resistance to control strategies that depend on insecticides and Bt toxins in transgenic plants. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. Researchers from USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL in collaboration with colleagues at the University of Florida, examined gene editing as a method to modify the genome of the fall armyworm as a tool to examine the genetics of resistance mechanisms. The abdominal-A gene of the fall armyworm was targeted for modification in this pest moth. Gene edited larvae and adult moths were found that showed malformations consistent with gene editing activity. The mutant moths were sterile. DNA sequencing of these mutant moths showed that gene editing was successful and had created deletions in the targeted gene. These results demonstrate that the gene editing system is highly efficient in modifying the fall armyworm genome and can potentially be used to validate gene functions necessary for an understanding of toxin resistance and help in the development of novel pest management approaches.

Technical Abstract: The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is an important pest of maize in the Americas and has recently been introduced into Africa. Fall armyworm populations in the field have developed resistance to control strategies that depend on insecticides and transgenic plants expressing Bacillus thuringiensis toxins. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. In the current study, we explored the possibility of using the CRISPR/Cas9 system to modify the genome of FAW. We first identified and characterized the abdominal-A (Sfabd-A) gene of FAW. Sfabd-A single guide RNA (sgRNA) and Cas9 protein were injected into 244 embryos of FAW. Sixty-two embryos injected with Sfabd-A guide RNA hatched. Of these hatched embryos, twelve developed into larvae that displayed typical aba-A mutant phenotypes such as fused segments. Of the twelve mutant larvae, three and five eventually developed into female and male moths, respectively. Most mutant moths were sterile, and one female produced a few unviable eggs when it was outcrossed to a wild-type male. Genotyping of 20 unhatched Sfabd-A sgRNA-treated embryos and 42 moths that developed from Sfabd-A sgRNA-treated embryos showed that 100% of the unhatched embryos and 50% of the moths contained indel mutations at the abd-A genomic locus near the guide RNA target site. These results suggest that the CRISPR/Cas9 system is highly efficient in editing FAW genome and can potentially be used to validate gene function to facilitate an understanding of resistance evolution and development of novel pest management approaches.