۱- Introduction

۲- Material and methods

۳- Results

۴- Discussion

References

Abstract

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.

Introduction

Mites and ticks are members of the chelicerates, the largest group of terrestrial animals after insects. The two-spotted spider mite, T. urticae, and other spider mites are important crop pests worldwide. This herbivore species is at the extreme end of the generalist-to specialist spectrum and can feed on a staggering 1,100 plant species. Not surprisingly, it is currently reported as the ‘most resistant’ pest worldwide, as it developed resistance to more than 90 acaricides (Mota-Sanchez and Wise, 2019; Van Leeuwen and Dermauw, 2016; Van Leeuwen et al., 2015). In 2011, a 90 Mb high-quality Sanger-sequenced genome became available for this species (Grbic et al., 2011). This allowed to disentangle some of the molecular mechanisms underlying resistance, whether to man-made pesticides or plant secondary compounds. The extreme adaptation potential of T. urticae was associated with specific gene expansions in known detoxification enzyme families, such as cytochrome P450 monooxygenases, glutathione-S-transferases, carboxyl-choline esterases, an unexpected repertoire of ABC and MFS transporters, and a proliferation of cysteine peptidases (Dermauw et al., 2013a; Dermauw et al., 2013b; Grbic et al., 2011; Santamaría et al., 2012).