CRISPR/Cas9 mediated editing of pheromone biosynthesis activating neuropeptide (PBAN) gene disrupts mating in the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

Abstract

The Fall armyworm, Spodoptera frugiperda, is a globally important invasive pest, primarily on corn, causing severe yield loss. Overuse of synthetic chemicals has caused significant ecological harm, and in many instances control has failed. Therefore, developing efficient, environmentally friendly substitutes for sustainable management of this pest is of high priority. CRISPR/Cas9-mediated gene editing causes site-specific mutations that typically result in loss-of-function of the target gene. In this regard, identifying key genes that govern the reproduction of S. frugiperda and finding ways to introduce mutations in the key genes is very important for successfully managing this pest. In this study, the pheromone biosynthesis activator neuropeptide (PBAN) gene of S. frugiperda was cloned and tested for its function via a loss-of-function approach using CRISPR/Cas9. Ribonucleoprotein (RNP) complex (single guide RNA (sgRNA) targeting the PBAN gene + Cas9 protein) was validated through in vitro restriction assay followed by embryonic microinjection into the G0 stage for in vivo editing of the target gene. Specific suppression of PBAN by CRISPR/Cas9 in females significantly affected mating. Mating studies between wild males and mutant females resulted in no fecundity. This was in contrast to when mutant males were crossed with wild females, which resulted in reduced fecundity. These results suggest that mating disruption is more robust where PBAN is edited in females. The behavioural bioassay using an olfactometer revealed that mutant females were less attractive to wild males compared to wild females. This study is the first of its kind, supporting CRISPR/Cas9 mediating editing of the PBAN gene disrupting mating in S. frugiperda. Understanding the potential use of these molecular techniques may help develop novel management strategies that target other key functional genes.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-023-03798-3.

Keywords: CRISPR/Cas9; Genome editing; Mating disruption; PBAN; Spodoptera frugiperda.

Fig. 1

Gene structure of S. frugiperda PBAN. S. frugiperda PBAN contains six exons (grey boxes) and five introns (black lines). The mRNA and sgRNA sequences in blue colour text are represented above and below the gene structure, respectively. The red colour bar in exon 4 represents the position of sgRNA. The red colour arrow represents the cut site, which is three nucleotides upstream from the PAM (represented inside the yellow colour arrow)

Fig. 2

In vitro digestion of S. frugiperda PBAN by RNP (sgRNA + Cas9) complex. L1- S. frugiperda PBAN amplicon, L2, L3, and L4 shows in vitro digestion assay of S. frugiperda PBAN amplicon with sgRNA1, sgRNA2 and sgRNA3, respectively. Only sgRNA1 shows in vitro cleavage, but sgRNA2 and sgRNA3 did not work. L5-100 base pair ladders

Fig. 3

Chromatographs and discordance outputs from the ICE (Inference of CRISPR Edits) software for a guide targeting the S. frugiperda PBAN gene. a Mutant 1 ♀ b Mutant 2 ♀ c Mutant 3 ♂. On the control trace, the guide RNA sequence is underlined in black, and the PAM site is underlined with a red dotted line. The cut site is indicated with a vertical dotted line on both control and edited traces. The histogram on the right shows the percentage of each indel type in the sample. On the left, a discordance plot displays the level of alignment between the mutant (green) and wild (orange) traces before and after the cut site (vertical black dotted line). Typically, the mutant and wild traces are aligned before the cut site and then become discordant after the cut site. Finally, the relative contribution of each sequence with INDELs, the per cent contribution and edited sequences were given

Fig. 3

Chromatographs and discordance outputs from the ICE (Inference of CRISPR Edits) software for a guide targeting the S. frugiperda PBAN gene. a Mutant 1 ♀ b Mutant 2 ♀ c Mutant 3 ♂. On the control trace, the guide RNA sequence is underlined in black, and the PAM site is underlined with a red dotted line. The cut site is indicated with a vertical dotted line on both control and edited traces. The histogram on the right shows the percentage of each indel type in the sample. On the left, a discordance plot displays the level of alignment between the mutant (green) and wild (orange) traces before and after the cut site (vertical black dotted line). Typically, the mutant and wild traces are aligned before the cut site and then become discordant after the cut site. Finally, the relative contribution of each sequence with INDELs, the per cent contribution and edited sequences were given

Fig. 3

Chromatographs and discordance outputs from the ICE (Inference of CRISPR Edits) software for a guide targeting the S. frugiperda PBAN gene. a Mutant 1 ♀ b Mutant 2 ♀ c Mutant 3 ♂. On the control trace, the guide RNA sequence is underlined in black, and the PAM site is underlined with a red dotted line. The cut site is indicated with a vertical dotted line on both control and edited traces. The histogram on the right shows the percentage of each indel type in the sample. On the left, a discordance plot displays the level of alignment between the mutant (green) and wild (orange) traces before and after the cut site (vertical black dotted line). Typically, the mutant and wild traces are aligned before the cut site and then become discordant after the cut site. Finally, the relative contribution of each sequence with INDELs, the per cent contribution and edited sequences were given

Fig. 4

Homology models representing the changes in S. frugiperda PBAN protein structure in mutants compared with the wild. The position of sgRNA is represented in red colour, and corresponding amino acids were marked in black colour text inside the zoomed portion in the PBAN protein structure of a wild insect. The altered PBAN protein structure of three mutant individuals was also represented with the sgRNA position marked in red colour

Fig. 5

The effect of CRISPR/Cas9 induced mutation of S. frugiperda PBAN on the mating behaviors of males with knockout mutant females

Fig. 6

The effect of CRISPR/Cas9 induced mutation of PBAN on the fecundity rate and hatching percent of S. frugiperda adults. Wild (un-injected) male and female were crossed for control