CRISPR/Cas9 mediates efficient site‐specific mutagenesis of the odorant receptor co‐receptor (Orco) in the malaria vector Anopheles sinensis

Abstract

BACKGROUND: Anopheles sinensis is the most widely distributed mosquito species and is the main transmitter of Plasmodium vivax malaria in China. Most previous research has focused on the mechanistic understanding of biological processes in An. sinensis and novel ways of interrupting malaria transmission. However, the development of functional genomics and genetics-based vector control strategies against An. sinensis remain limited because of insufficient site-specific genome editing tools.RESULTS: We report the first successful application of the CRISPR/Cas9 mediated knock-in for highly efficient, site-specific mutagenesis in An. sinensis. The EGFP marker gene driven by the 3 × P3 promoter was precisely integrated into the odorant receptor co-receptor (Orco) by direct injections of Cas9 protein, double-stranded DNA donor, and Orco-gRNA. We achieved a mutation rate of 3.77%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed by polymerase chain reaction (PCR) amplification and sequencing. The Orco mutation severely impaired mosquito sensitivity to some odors and their ability to locate and discriminate a human host.CONCLUSION: Orco was confirmed as a key mediator of multiple olfactory-driven behaviors in the An. sinensis life cycle, highlighting the importance of Orco as a key molecular target for malaria control. The results also demonstrated that CRISPR/Cas9 was a simple and highly efficient genome editing technique for An. sinensis and could be used to develop genetic control tools for this vector.

Genome-wide identification of candidate chemosensory receptors in the bean bug Riptortus pedestris (Hemiptera: Alydidae) and the functional verification of its odorant receptor co-receptor (Orco) in recognizing aggregation pheromone

“…For example, RNAi-based silencing of the Orco gene in Protaetia brevitarsis adults significantly impaired their location of aggregation pheromones and food sources (Zhang et al, 2021). Knocking out of the Orco gene through CRISPR-Cas9 seriously affects the olfactory-related behaviors as well, which has been confirmed in Anopheles coluzzii (Sun Y. et al, 2020), A. sinensis (Wang et al, 2022), Bactrocera dorsalis (Xu et al, 2022), B. mori (Liu et al, 2017), Harpegnathos saltator (Yan et al, 2017), Helicoverpa armigera (Fan et al, 2022), Locusta migratoria (Li et al, 2016), Spodoptera frugiperda (Sun et al, 2023) and S. littoralis (Koutroumpa et al, 2016). Our results of silencing RpedOrco in R. pedestris reached the similar conclusion.…”

“…In light of its necessity in olfactory recognition, Orco attracts increasing focus as a target for developing pest control agents (Fan et al, 2022). To date, mutants of many insect species have been generated to characterize and investigate the function of Orco through gene silencing with RNA interference (RNAi) or gene editing with CRISPR-Cas9 (Tateishi et al, 2022;Wang et al, 2022). For example, RNAi-based silencing of the Orco gene in Protaetia brevitarsis adults significantly impaired their location of aggregation pheromones and food sources (Zhang et al, 2021).…”

Innovative Methods of Mosquito Management

“…As an alternative to sterilizing males for the purpose of insect population control, genetic engineering is often used to introduce a gene into mosquito vectors that causes them to die off on their own [80]. It was discovered that OX513A males in Malaysia had similar lifespans [81] and spreading capacities, however the most recent production of OX513A males in Brazil ended in a large drop of the target wild population.…”