A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster

Abstract

Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.

Keywords: CRISPR; Insecticide resistance; Nicotinic acetylcholine receptor; Spinosad; nAChR.

Graphical abstract

Fig. 1

Dα6 exon organisation, gRNA target site and HDR template. A) Exon organisation, white arrows indicating alternative exon 3a/b and 8a/b (top) and 20 nt CRISPR/Cas9 target site as indicated by the large arrow above the sequence followed by the ‘ngg’ PAM motif (bottom). B) Nucleotide alignment of the 110 nt HDR template with Dα6. The single C/G substitution prevents re-cleavage of Cas9 through a mismatch in the gRNA seed sequence (last 12 nt of gRNA) and the double substitution GC/AA introduces a codon substitution from glycine (G) to glutamatic acid (E).

Fig. 2

Direct sequencing of Dα6 PCR fragments amplified from gDNA isolated from non-modified flies (wildtype), and spinosad resistant progeny after CRISPR (KO = knock outs, Mut = precise point mutation). The gRNA target site and exon 9 are annotated in purple and grey respectively. Point mutations are highlighted with orange arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Direct sequencing of Dα6 PCR fragments amplified from cDNA pools isolated from non-modified flies (lig4 KO Cas9), and flies carrying the G275E mutation (CRISPR+) codon position 901–903. Exons 8 and 9 are annotated in grey.

Fig. 4

Non-linear log dose-response plots for spinosad against Drosophila melanogaster strains and F1 progeny. Error bars represent standard deviation. A) Canton-S, lig4 KO Cas9, Dα6 G275E and F1 progeny of Dα6 G275E × Canton-S and Dα6 G275E × lig4 KO Cas9 respectively. B) Canton-S, lig4 KO Cas9, Dα6 KO and F1 progeny of Dα6 KO × Canton-S and Dα6 KO × lig4 KO Cas9 respectively.

Fig. 5

Amino acid sequence alignment of F. occidentalis nAChR α6 and D. melanogaster nAChR α6 in variant exon 3b/8a. G275E is indicated by an orange arrow and counted from the first amino acid (G) after the putative signal peptide indicated by a pink arrow. Transmembrane regions TM1-4 are annotated with red arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)