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CRISPR/Cas9, efficient in inducing a loss of olfaction in a crop pest insect

CRISPR/Cas9, interesting opportunities to study the function of genes of interest in crop pest

Spodoptera littoralis. © INRA
Updated on 06/08/2017
Published on 11/30/2016

Lepidoptera account for more than 10% of all the species described to date. They include some redoubtable crop pests that are sometimes difficult to study because of a lack of genetic techniques appropriate for use in these insects.

But the development of genome editing techniques has recently seen extraordinary growth. Efficient and rapid, they can induce specific breaks in double-strand DNA, offering fascinating perspectives for the study of genomes. Seduced by these arguments, INRA scientists and their colleagues at the Natural History Museum have studied the feasibility of their use in the cotton leafworm, Spodoptera littoralis, which is a dangerous crop pest.

CRISPR/Cas9 in insects, where technical prowess is a prerequisite

The scientists used the CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats Associated protein 9) system. In practice, the CRISPR/Cas9 machinery is an enzyme and its guide is a short RNA sequence. If the DNA of interest is related to the guide sequence, then the CRISPR/Cas9 machinery recognises and cuts it. This triggers the natural DNA repair mechanism that is able to introduce errors into the DNA sequence it is repairing, and thus totally inactive a gene.

It is therefore necessary to design this machinery and then introduce it at an early stage into an insect’s germ cells, before selecting and then analysing the individuals carrying mutations. This is what the scientists have achieved by targeting the Orco gene, which is well known for its role in olfactory communication between insects; their aim was to explore its function in S. littoralis. It should be remembered that when pest insects (including S. littoralis) attack, they deploy specific behaviours that are closely linked to their olfactory capacities: reproduction, recognition and the choice of the host plant and laying sites, etc.

Thus 700 larvae were born from the 2570 eggs into which the scientists had previously injected Cas9 and its guide. It should be pointed out that the sequence of this guide had been designed specifically to recognize part of the genome sequence of the Orco gene in S. littoralis. Genetic analysis and functional investigations then made it possible for the research team to characterise the mutations presented by the individuals and their progeny.

Almost all the caterpillars born from the injected eggs (90%) displayed mutations in the Orco gene. Most of these mutations (70%) had been transmitted sexually to the progeny.

CRISPR/Cas9 in S. littoralis, or how to induce anosmia in a crop pest

The individuals presenting harmful mutations in their Orco gene had also lost their ability to smell a series of odorant compounds from plants, and the sex pheromones of females. Everything suggested that in this butterfly, the olfactory receptors for these odours functioned by interacting with the Orco co-receptor, as had indeed been demonstrated previously in the model fruit fly Drosophila melanogaster.

On the other hand, the same individuals continued to perceive the odour of propionic acid, which is emitted during the decomposition of plants, thus showing that the detection of such products is assured by other receptors that are independent of any interaction with the Orco co-receptor.

All these findings testify to the actual transformation of sex cells and the stable transmission of mutations; more generally, they reveal the considerable efficiency of the CRISPR/Cas9 system used during this study to target a gene (in this case, the Orco gene in S. littoralis) in order to induce mutations.

Potentially applicable to other Lepidoptera species, the CRISPR/Cas9 technique offers interesting opportunities to study the function of genes of interest in non-model insects such as crop pests. This technology will clearly benefit from concomitant advances in the high-throughput sequencing of genomes, which suggests that in the longer term, it may be possible to develop new technologies for the control of crop insect pests.

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Plant Health and Environment
Associated Centre(s):
Versailles-Grignon

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Koutroumpa F.A., Monsempes C., François M.C., de Cian A., Royer C., Concordet J.P., Jacquin-Joly E. (2016) Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth. Sci. Reports 6:29620 | DOI: 10.1038/srep29620

Orco, a short explanation

Communication is vital for survival and reproduction in insects, which use their senses to perceive their environment and fellow creatures
Chemical communication, in the form of odours perceived by olfaction, involves specific sensory receptors, the olfactory receptors (OR). In order to function, these receptors associate themselves with a co-receptor (that is always the same) called Orco. If mutations target Orco, most odorant signals are no longer detected by the olfactory receptors, and the insect becomes anosmic – it no longer has a sense of smell.