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In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Invasive insect pests, currently, pose a serious economic threat to several staple crops all over the world, one such being the fall armyworm, Spodoptera frugiperda. It was first observed in Africa since 2016, outside of its natural habitat in the Americas. Subsequently, it invaded several countries in South and South East Asia and also very recently in Australia. In all the newly invaded regions, maize is the principal crop attacked causing a serious economic concern to the poor farmers, particularly in the developing countries. Owing to the innate genetic ability, it defies many of the management options that include insecticides, Bt transgenics, and so forth. This is due to its high mobility, polyphagy and ability for quick development of resistance to several classes of insecticides. At this critical juncture, CRISPR/Cas9 mediated genome editing has shown a lot of promise in developing a novel area-wide pest management strategy called precision-guided sterile insect technique (pgSIT). pgSIT was initially demonstrated in Drosophila melanogaster which holds a greater promise for the environmentally friendly management of several globally significant agricultural pests such as S. frugiperda. Therefore, before developing both sgRNA and Cas9 transgenic lines, we have validated the target gene such as tssk2 through a non-transgenic approach by microinjecting ribo nucleo protein complex (Cas9 protein and tssk2 sgRNA) into G 0 eggs of S. frugiperda. In the current investigation, we have obtained five edited males with distinct mutations which were further used for crossing studies to ascertain the effect of tssk2 editing affecting egg hatchability., (© 2024 Wiley Periodicals LLC.)