A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster.

Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off-target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5'-NG-3' PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome-wide off-target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome-editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non-NGG PAM site TGA, xCas9 could produce DNA cleavage and indel-mediated disruption on the target gene. However, for other non-NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.

Characterization of three serotonin receptors from the small white butterfly, Pieris rapae.

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT1A, Piera5-HT1B, Piera5-HT7) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT1A or Piera5-HT1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT7 with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT1B and Piera5-HT7 respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects.