Amezian, D.; De Graeve, F.; Mettumpurath Sasi, R.; Vanhaecht, L.; Mocchetti, A.; Villacis-Perez, E.; Kant, M. R.; De Rouck, S.; Van Leeuwen, T.

Tetranychus evansi is an invasive spider mite pest of solanaceous crops worldwide. Its global expansion and ability to rapidly develop acaricide resistance highlight the need for robust genomic resources and functional genetic tools to design custom control strategies. Here, we deliver a high-quality genome assembly and establish efficient CRISPR/Cas9 editing in T. evansi to enable mechanistic studies of host adaptation and pesticide resistance. Using an inbred line and Oxford Nanopore Technologies (ONT) long-read sequencing (~36x), we assembled an 89 Mb genome into 13 contigs (N50 = 18.6 Mb) with high completeness and annotated 14,246 protein-coding genes. We manually curated the principal detoxification gene families (P450s, CCEs, GSTs, UGTs, ABC transporters and DOGs), revealing consistently smaller repertoires than in the highly polyphagous relative Tetranychus urticae. To enable reverse genetics, we adapted SYNCAS (saponin + branched amphiphilic peptide capsules) for maternal delivery of CRISPR/Cas9 in T. evansi. Targeting the phytoene desaturase (PD) pigmentation marker produced reliable knockouts with visible albinism and mean editing efficiencies of ~9.6-14.7%, allowing establishment of stable mutant lines. We further applied precision gene editing to knock-in (KI) the M918T and M918L substitutions into the voltage-gated sodium channel (VGSC). While M918L was lethal in T. evansi, we generated multiple homozygous lines for M918T (mean KI ~4.4%). Bioassays demonstrated that while the mutation caused extremely high levels of bifenthrin resistance (RR>1000), this was less so for cyfluthrin (RR=164), revealing the independent and specific role of M918T in pyrethroid resistance. Collectively, these resources establish T. evansi as a tractable system for reverse genetic analysis and provide a reference for future comparative and population genomics.